Franka Klatte-Schulz

Do Patient Age and Sex Influence Tendon Cell Biology and Clinical/Radiographic Outcomes After Rotator Cuff Repair?

Background: Many clinical and radiographic studies suggest that patient age and sex have an influence on rotator cuff (RC) repair outcomes. However, these findings result from retrospective statistical analyses and cannot provide a causal answer. Purpose: To analyze whether age and sex influence the biological potential at the time of RC repair or midterm clinical and radiographic outcomes. Also assessed was the effect of the biological potential on intraindividual clinical/radiographic results. Study Design: Cohort study; Level of evidence, 2. Methods: A total of 40 patients underwent arthroscopic RC repair. At the time of surgery (t = 0), supraspinatus tendon biopsy specimens were obtained, cultivated, and assessed for their biological potential, particularly (1) cell growth and (2) collagen type I production. After a follow-up at 24 months (t = 1), all patients were assessed by clinical scores (Constant score, subjective shoulder value, American Shoulder and Elbow Surgeons [ASES] score, and Western Ontario Rotator Cuff Index [WORC] score) and underwent magnetic resonance imaging to determine RC integrity. The data were examined for age- and sex-related differences and to identify the correlation between biological potential (t = 0) and clinical/radiographic outcome (t = 1). Results: The follow-up rate for the imaging and clinical evaluation was 100%. Age, but not sex, influenced the biological tendon cell parameters at t = 0. However, there was no effect of age or sex on the clinical and radiographic results at t = 1. Furthermore, no correlation was observed between the initial biological parameters and later clinical outcomes or radiographic RC integrity. Finally, there was no significant difference between intact and nonhealed repairs in terms of the respective clinical scores. Conclusion: Age, but not sex, was found to have a negative effect on RC tendon cell biology. However, neither sex nor, in particular, a higher age influenced repair outcomes after 24 months.

Characteristics and Stimulation Potential with BMP-2 and BMP-7 of Tenocyte-Like Cells Isolated from the Rotator Cuff of Female Donors

Tendon bone healing of the rotator cuff is often associated with non-healing or recurrent defects, which seems to be influenced by the patient’s age and sex. The present study aims to examine cellular biological characteristics of tenocyte-like cells that may contribute to this impaired rotator cuff healing. Moreover, a therapeutic approach using growth factors could possibly stimulate tendon bone healing. Therefore, our second aim was to identify patient groups who would particularly benefit from growth factor stimulation. Tenocyte-like cells isolated from supraspinatus tendons of female donors younger and older than 65 years of age were characterized with respect to different cellular biological parameters, such as cell density, cell count, marker expression, collagen-I protein synthesis, and stem cell potential. Furthermore, cells of the donor groups were stimulated with BMP-2 and BMP-7 (200 and 1000 ng/ml) in 3D-culture and analyzed for cell count, marker expression and collagen-I protein synthesis. Female donors older than 65 years of age showed significantly decreased cell count and collagen-I protein synthesis compared to cells from donors younger than 65 years. Cellular biological parameters including cell count, collagen-I and –III expression, and collagen-I protein synthesis of cells from both donor groups were stimulated with BMP-2 and BMP-7. The cells from donors older than 65 years revealed a decreased stimulation potential for cell count compared to the younger group. Cells from female donors older than 65 years of age showed inferior cellular biological characteristics. This may be one reason for a weaker healing potential observed in older female patients and should be taken into consideration for tendon bone healing of the rotator cuff.

Relationship between muscle fatty infiltration and the biological characteristics and stimulation potential of tenocytes from rotator cuff tears

The healing after rotator cuff surgery is still dissatisfying, and increased muscle fatty infiltration even more impairs the healing success. To achieve sufficient healing after rotator cuff reconstructions, the use of growth factors may be one possibility. The aim of the study was to identify a possible relationship between fatty infiltration of the supraspinatus muscle and cellular biological characteristics and stimulation potential of tenocyte‐like cells (TLCs). TLCs of 3 donor groups differing in grade of muscle fatty infiltration were analyzed for their cellular characteristics and were stimulated with BMP‐2 or BMP‐7 in a 3D scaffold culture. The cell count and potency for self‐renewal were significantly decreased in TLCs from donors with high muscle fatty infiltration compared to the lower fatty infiltration groups. Cell count and collagen‐I expression as well as protein synthesis were stimulated by growth factors. Interestingly, TLCs of the high fatty infiltration group exhibited a weaker stimulation potential compared to the other groups. TLCs from donors with high muscle fatty infiltration generally revealed inferior characteristics compared to cells of lower fatty infiltration groups, which may be one reason for a weaker healing potential and may represent a possible starting point for the development of future treatment options.

Do Matrix Metalloproteases and Tissue Inhibitors of Metalloproteases in Tenocytes of the Rotator Cuff Differ with Varying Donor Characteristics

An imbalance between matrix metalloproteases (MMPs) and the tissue inhibitors of metalloproteases (TIMPs) may have a negative impact on the healing of rotator cuff tears. The aim of the project was to assess a possible relationship between clinical and radiographic characteristics of patients such as the age, sex, as well as the degenerative status of the tendon and the MMPs and TIMPs in their tenocyte-like cells (TLCs). TLCs were isolated from ruptured supraspinatus tendons and quantitative Real-Time PCR and ELISA was performed to analyze the expression and secretion of MMPs and TIMPs. In the present study, MMPs, mostly gelatinases and collagenases such as MMP-2, -9 and -13 showed an increased expression and protein secretion in TLCs of donors with higher age or degenerative status of the tendon. Furthermore, the expression and secretion of TIMP-1, -2 and -3 was enhanced with age, muscle fatty infiltration and tear size. The interaction between MMPs and TIMPs is a complex process, since TIMPs are not only inhibitors, but also activators of MMPs. This study shows that MMPs and TIMPs might play an important role in degenerative tendon pathologies.

Comparative Analysis of Different Platelet Lysates and Platelet Rich Preparations to Stimulate Tendon Cell Biology: An In Vitro Study

The poor healing potential of tendons is still a clinical problem, and the use of Platelet Rich Plasma (PRP) was hypothesized to stimulate healing. As the efficacy of PRPs remains unproven, platelet lysate (PL) could be an alternative with its main advantages of storage and characterization before use. Five different blood products were prepared from 16 male donors: human serum, two PRPs (Arthrex, (PRP-ACP); RegenLab (PRP-BCT)), platelet concentrate (apheresis, PC), and PL (freezing-thawing destruction of PC). Additionally, ten commercial allogenic PLs (AlloPL) from pooled donors were tested. The highest concentration of most growth factors was found in AlloPL, whereas the release of growth factors lasted longer in the other products. PRP-ACP, PRP-BCT, and PC significantly increased cell viability of human tenocyte-like cells, whereas PC and AlloPL increased Col1A1 expression and PRP-BCT increased Col3A1 expression. MMP-1, IL-1β, and HGF expression was significantly increased and Scleraxis expression decreased by most blood products. COX1 expression significantly decreased by PC and AlloPL. No clear positive effects on tendon cell biology could be shown, which might partially explain the weak outcome results in clinical practice. Pooled PL seemed to have the most beneficial effects and might be the future in using blood products for tendon tissue regeneration.

Time-Dependent Alterations of MMPs, TIMPs and Tendon Structure in Human Achilles Tendons after Acute Rupture

A balance between matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) is required to maintain tendon homeostasis. Variation in this balance over time might impact on the success of tendon healing. This study aimed to analyze structural changes and the expression profile of MMPs and TIMPs in human Achilles tendons at different time-points after rupture. Biopsies from 37 patients with acute Achilles tendon rupture were taken at surgery and grouped according to time after rupture: early (2–4 days), middle (5–6 days), and late (≥7 days), and intact Achilles tendons served as control. The histological score increased from the early to the late time-point after rupture, indicating the progression towards a more degenerative status. In comparison to intact tendons, qRT-PCR analysis revealed a significantly increased expression of MMP-1, -2, -13, TIMP-1, COL1A1, and COL3A1 in ruptured tendons, whereas TIMP-3 decreased. Comparing the changes over time post rupture, the expression of MMP-9, -13, and COL1A1 significantly increased, whereas MMP-3 and -10 expression decreased. TIMP expression was not significantly altered over time. MMP staining by immunohistochemistry was positive in the ruptured tendons exemplarily analyzed from early and late time-points. The study demonstrates a pivotal contribution of all investigated MMPs and TIMP-1, but a minor role of TIMP-2, -3, and -4, in the early human tendon healing process.

An investigation of BMP-7 mediated alterations to BMP signalling components in human tenocyte-like cells

The incidence of tendon re-tears post-surgery is an ever present complication. It is suggested that the application of biological factors, such as bone morphogenetic protein 7 (BMP-7), can reduce complication rates by promoting tenogenic characteristics in in vitro studies. However, there remains a dearth of information in regards to the mechanisms of BMP-7 signalling in tenocytes. Using primary human tenocyte-like cells (hTLCs) from the supraspinatus tendon the BMP-7 signalling pathway was investigated: induction of the BMP associated Smad pathway and non-Smad pathways (AKT, p38, ERK1/2 and JNK); alterations in gene expression of BMP-7 associated receptors, Smad pathway components, Smad target gene (ID1) and tenogenic marker scleraxis. BMP-7 increases the expression of specific BMP associated receptors, BMPR-Ib and BMPR-II, and Smad8. Additionally, BMP-7 activates significantly Smad1/5/8 and slightly p38 pathways as indicated by an increase in phosphorylation and proven by inhibition experiments, where p-ERK1/2 and p-JNK pathways remain mainly unresponsive. Furthermore, BMP-7 increases the expression of the Smad target gene ID1, and the tendon specific transcription factor scleraxis. The study shows that tenocyte-like cells undergo primarily Smad8 and p38 signalling after BMP-7 stimulation. The up-regulation of tendon related marker genes and matrix proteins such as Smad8/9, scleraxis and collagen I might lead to positive effects of BMP-7 treatment for rotator cuff repair, without significant induction of osteogenic and chondrogenic markers.

Different Achilles Tendon Pathologies Show Distinct Histological and Molecular Characteristics

Reasons for the development of chronic tendon pathologies are still under debate and more basic knowledge is needed about the different diseases. The aim of the present study was therefore to characterize different acute and chronic Achilles tendon disorders. Achilles tendon samples from patients with chronic tendinopathy (n = 7), chronic ruptures (n = 6), acute ruptures (n = 13), and intact tendons (n = 4) were analyzed. The histological score investigating pathological changes was significantly increased in tendinopathy and chronic ruptures compared to acute ruptures. Inflammatory infiltration was detected by immunohistochemistry in all tendon pathology groups, but was significantly lower in tendinopathy compared to chronic ruptures. Quantitative real-time PCR (qRT-PCR) analysis revealed significantly altered expression of genes related to collagens and matrix modeling/remodeling (matrix metalloproteinases, tissue inhibitors of metalloproteinases) in tendinopathy and chronic ruptures compared to intact tendons and/or acute ruptures. In all three tendon pathology groups markers of inflammation (interleukin (IL) 1β, tumor necrosis factor α, IL6, IL10, IL33, soluble ST2, transforming growth factor β1, cyclooxygenase 2), inflammatory cells (cluster of differentaition (CD) 3, CD68, CD80, CD206), fat metabolism (fatty acid binding protein 4, peroxisome proliferator-activated receptor γ, CCAAT/enhancer-binding protein α, adiponectin), and innervation (protein gene product 9.5, growth associated protein 43, macrophage migration inhibitory factor) were detectable, but only in acute ruptures significantly regulated compared to intact tendons. The study gives an insight into structural and molecular changes of pathological processes in tendons and might be used to identify targets for future therapy of tendon pathologies.

51 Insights Into Bmp-7 Signalling Of Rotator Cuff Tenocytes

Introduction We reported previously that important cellular characteristics such as the cell activity and the expression and synthesis of collagen I can be strongly enhanced by stimulation of tenocytes with BMP-7 [Klatte-Schulz et al. 2014]. Since tendon healing after surgical reconstructions still represents a challenging topic, due to complications such as retears or non-healing of the tendon tissue, the augmentation of the healing using growth factors like BMP-7 may be a possible treatment option. What remains unknown is how BMP-7 signals in tenocytes, which was analysed in the present study. Methods Tenocytes of 6 male donors (63–69 years) isolated from biopsies of supraspinatus tendon tears were seeded in cell culture dishes, cultured for 3–5 days and stimulated with 200 or 1000 ng/ml rhBMP-7. Protein lysate samples were collected after 15, 30, 60 and 120 min and the phosphorylation of Smad1/5/8 was analysed using Western Blotting technique. RNA was isolated after 1, 2, 4 and 8 h from the cells. The gene expression of BMP-Receptors (BMPR-Ia, BMPR-Ib, BMPRII), Activin receptors (ActR-I, ActR-IIa, ActR-IIb), Smad 1, 4, 5, 8 and the BMP target gene ID1, as well as scleraxis, osteocalcin and collagen II was analysed by Real-Time PCR. Statistics: Mann-Whitney-U Test, p ≤ 0.05, Bonferroni Holm Correction Results The relative phosphorylation level of Smad1/5/8 was significantly increased 30 and 60 min after stimulation with BMP-7 compared to unstimulated control cells (Figure 1). Abstract 51 Figure 1 Smad phosphorylation relative to total Smad and GAPDH and normalized to unstimulated control The expression of BMPR-Ib was increased the most compared to both other type I receptors 4 and 8 h after stimulation. In the type II receptor group, BMPR-II showed the highest increase after 2 to 8 h, while ActR-IIb was down-regulated after 2 h and 4 h. The expression of Smad1 and 4 was slightly increased and Smad5 showed no increase of expression. Smad8 expression was strongly up-regulated 4 h and 8 h after stimulation (Figure 2). The expression of ID1 was increased already at 1 h and 2 h and strongly enhanced 8 h after stimulation. Scleraxis expression showed a strong increase at 1, 2 and 4 h and a massive increase at 8 h after stimulation. No change in the osteocalcin and collagen II expression was found after BMP-7 stimulation. Abstract 51 Figure 2 Smad8 expression relative to 18 s and normalised to control. Discussion The study showed that the type I receptor BMPR-Ib and the type II receptor BMPR-II seem to be the most regulated receptors on their mRNA level after tenocyte stimulation with BMP-7. Additionally, the receptor-regulated Smad8 seems to represent the essential signalling molecule in tenocytes after BMP-7 stimulation. An important role of Smad8 in tenogenic differentiation was reported previously. [Hoffmann et al., 2006] The strong increase of the tendon related gene scleraxis in the cells may underline the positive effect of BMP-7 treatment for rotator cuff repair. References Klatte-Schulz, et al. J Orthop Res. 2014;32(1):129–37 Hoffmann, et al. J Clin Invest. 2006;116(4):940–52