Johannes Zeichen

The Proliferative Response of Isolated Human Tendon Fibroblasts to Cyclic Biaxial Mechanical Strain

At the cellular level, dynamic strain plays a key role in cell stimulation and organization of the extracellular matrix. Although positive effects of physical strain on tendon tissue are well known, little knowledge exists on how mechanical strain affects tendon cells. In this study, human tendon fibroblasts from patellar tendon were cultured on silicone dishes. Subsequently, cyclic biaxial mechanical strain was applied to the dishes for 15, 30, and 60 minutes using a specially developed cell stretching system. After the fibroblasts were strained, cells were tested for proliferation at 6, 12, and 24 hours. As a control, cells were grown on silicone dishes but did not receive any strain. A biphasic response in proliferation was observed for the 15- and 60-minute strain periods: at 6 hours and 24 hours there was more proliferation than at 12 hours. After a strain duration time of 30 minutes, a lower proliferation rate was measured compared with control levels. This study shows that application of mechanical stress to tendon fibroblasts resulted in an alteration of cellular proliferation depending on the stress time. Our results may implicate future modifications in the treatment of ligament and tendon injuries.

Achilles Tendon and Paratendon Microcirculation in Midportion and Insertional Tendinopathy in Athletes

Background Neovascularisation can be detected qualitatively by Power Doppler in Achilles tendinopathy. Quantitative data regarding tendon microcirculation have not been established and may be substantial. Purpose To assess the microcirculation of the Achilles tendon and the paratendon in healthy volunteers as well as in athletes with either midportion or insertional tendinopathy. Study Design Cohort study; Level of evidence, 2. Methods In 66 physically active volunteers, parameters of Achilles tendon and paratendon microcirculation, such as tissue oxygen saturation, relative postcapillary venous filling pressures, and microcirculatory blood flow, were determined at rest at 2-mm and 8-mm tissue depths. Forty-one patients never had Achilles pain (25 men, 27 ± 8 years), 14 patients had insertional pain (7 men, 29 ± 8 years), and 11 patients had midportion tendinopathy (7 men, 38 ± 13 years, not significant). Results Achilles tendon diameter 2 cm and 6 cm proximal to the insertion was increased in symptomatic tendons. Compared with the uninvolved opposite tendon, deep microcirculatory blood flow was significantly elevated at insertional (160 ± 79 vs 132 ± 42, P<. 05) as well as in midportion tendinopathy (150 ± 74 vs 119 ± 34, P<. 05). The microcirculation in the uninvolved opposite tendon and the normal athlete controls were not significantly different from each other (132 ± 42 insertional asymptomatic vs 119 ± 34 mid-portion vs 120 ± 48 healthy tendon). Insertional paratendon deep microcirculatory flow was elevated in all groups, whereas tissue oxygen saturation and relative postcapillary venous filling pressures were not significantly different. Conclusion Microcirculatory blood flow is significantly elevated at the point of pain in insertional and midportion tendinopathy. Postcapillary venous filling pressures are increased at both the midportion Achilles tendon and the midportion paratendon, whereas tissue oxygen saturation is not different among the studied groups. We found no evidence of an abnormal microcirculation of the asymptomatic limb in Achilles tendinopathy.

Proprioception after rehabilitation and reconstruction in knees with deficiency of the anterior cruciate ligament : a prospective, longitudinal study

We assessed proprioception in the knee using the angle reproduction test in 20 healthy volunteers, ten patients with acute anterior instability and 20 patients with chronic anterior instability after reconstruction of the anterior cruciate ligament (ACL). In addition, the Lysholm-knee score, ligament laxity and patient satisfaction were determined. Acute trauma causes extensive damage to proprioception which is not restored by rehabilitation alone. Three months after operation, there remained a slight decrease in proprioception compared with the preoperative recordings, but six months after reconstruction, restoration of proprioception was seen near full extension and full flexion. In the mid-range position, proprioception was not restored. At follow-up, 3.7 +/- 0.3 years after reconstruction, there was further improvement of proprioception in the mid-range position. There was no difference between open and arthroscopic techniques. The highest correlation was found between proprioception and patient satisfaction. After reconstruction of the ACL reduced proprioception may explain the poor functional outcome in some patients, despite restoration of mechanical stability.

Influence of perfusion and cyclic compression on proliferation and differentiation of bone marrow stromal cells in 3-dimensional culture

Until now, there has been no in vitro model that duplicates the environment of bone marrow. The purpose of this study was to analyze proliferation and differentiation of human bone marrow stromal cells (hBMSC) under the influence of continuous perfusion and cyclic mechanical loading. hBMSC of seven individuals were harvested, grown in vitro, and combined. 10(6) hBMSC were seeded on a bovine spongiosa disc and incubated in a bioreactor system. Cell culture was continued using three different conditions: Continuous perfusion (group A), 10% cyclic compression at 0.5Hz (group B) and static controls (group C). After 24h, 1, 2, and 3 weeks, we determined cell proliferation (MTS-assay) and osteogenic differentiation (osteocalcin ELISA, Runx2 mRNA). Tenascin-C mRNA was quantified to exclude fibroblastic differentiation. In groups A and B, proliferation was enhanced after 2 weeks (48.6+/-19.6x10(3) (A) and 44.6+/-14.3 x 10(3) cells (B)) and after 3 weeks (46.6+/-15.1 x 10(3) (A) and 44.8+/-10.2 x 10(3) cells (B)) compared with controls (26.3+/-10.8 x 10(3) (2 weeks) and 17.1+/-6.5 x 10(3) cells (3 weeks), p<0.03). Runx2 mRNA was upregulated in both stimulated groups after 1, 2, and 3 weeks compared to control (group A, 1 week: 5.2+/-0.7-fold; p<0.01, 2 weeks: 4.4+/-1.9-fold; p<0.01, 3 weeks: 3.8+/-1.7-fold; p=0.013; group B, 1 week: 3.6+/-1.1-fold, p<0.01, 2 weeks: 4.2+/-2.2-fold, p<0.01; 3 weeks: 5.3+/-2.7-fold, p<0.01). hBMSC stimulated by cyclic compression expressed the highest amount of osteocalcin at all time points (1 week: 294.5+/-88.4 mg/g protein, 2 weeks: 294.4+/-73.3mg/g protein, 3 weeks: 293.1+/-83.6 mg/g protein, p0.03). The main stimulus for cell proliferation in a 3-dimensional culture of hBMSC is continuous perfusion whereas mechanical stimulation fosters osteogenic commitment of hBMSC. This study thereby contributes to the understanding of physical stimuli that influence hBMSC in a 3-dimensional cell culture system.

Outcome analysis following open rotator cuff repair. Early effectiveness validated using four different shoulder assessment scales.

Abstract Evaluation of upper extremity function after reconstructive surgery is increasingly important both to predict outcome and for the control of cost-effectiveness. Three validated, self-administered shoulder questionnaires were applied prospectively in 23 otherwise healthy patients with rotator cuff deficiency and correlated to the Constant-Murley Shoulder Score and a visual analogue scale for satisfaction. Seven women and 16 men with combined tears of supraspinatus and infraspinatus (mean age 55.3 ± 10.5 years, r/l: 14/9, follow-up 57.8 ± 15.7 weeks) were gathered prospectively and evaluated pre- and postoperatively with the American Shoulder and Elbow Surgeons (ASES) Shoulder Index, the Simple Shoulder Test (SST) and the Disabilities of the Arm, Shoulder and Hand Module (DASH questionnaire). Additionally, a visual analogue scale for satisfaction was employed. All four scores and the visual analogue scale revealed improvement at a statistically significant level (P < 0.01) after surgery. All questionnaires showed a significant correlation with the Constant-Murley Shoulder Score (ASES: r = 0.871, P < 0.01; DASH: r = –0.758, P < 0.01, SST: r = 0.494, P < 0.05, Pearson’s correlation coefficient). Taken together, all questionnaires were easy to apply, and reliable evaluation of shoulder function was possible with significant correlation to the Constant-Murley Shoulder Score postoperatively. The SST was easy to apply, and compound outcome analysis was possible with the ASES Shoulder Index and DASH questionnaire. The DASH scale was the most complex evaluation instrument. The Constant-Murley Shoulder Score comprises a physical examination, which is advantageous but restricts the application to the office. For postoperative assessment without the patient having to return to the clinic, the ASES Shoulder Index is preferred because of its good correlation to the Constant-Murley Shoulder Score (r = 0.871) and the visual analogue scale for satisfaction (r = 0.762).

Modulation of cell functions of human tendon fibroblasts by different repetitive cyclic mechanical stress patterns.

Mechanical stress is a factor that is thought to play an essential role in tissue generation and reparation processes. The aim of the present study was to investigate the influence of different repetitive cyclic longitudinal stress patterns on proliferation, apoptosis and expression of heat shock protein (HSP) 72. To perform this study, human tendon fibroblasts were seeded on flexible silicone dishes. After adherence to the dish, cells were longitudinally stressed with three different repetitive stress patterns having a frequency of 1 Hz and an amplitude of 5%. The proliferation and apoptosis rates were investigated 0, 6, 12 and 24 hours after application of cyclic mechanical longitudinal strain. Expression of HSP 72 was tested after 0, 2, 4 and 8 hours. Control cells were also grown on silicone dishes, but did not receive any stress. Stress patterns applied during one day resulted in a significant increase in proliferation and a slight increase in apoptosis. HSP 72 expression was rather unchanged. A stress pattern applied during two days resulted in a reduced proliferation and apoptosis rate whereas the expression of HSP 72 showed a significant increase. This study shows that different stress patterns result in different cellular reactions dependent on the strength of applied stress. Repetitive stress applied during one day stimulated proliferation and apoptosis in contrast to an extended stress duration. The latter induced an inhibition of proliferation and apoptosis probably through an increased HSP 72 activity. This may be related to an excess of applied stress. Our results may implicate future modulation techniques for tissue reparation and tissue engineering.

α-Smooth muscle actin containing contractile fibroblastic cells in human knee arthrofibrosis tissue

IntroductionPrimary arthrofibrosis is of major concern after joint trauma or knee ligament surgery. The underlying mechanism in detail remains unclear. Highly differentiated fibroblastic cells, so-called myofibroblasts, express the actin isoform α-smooth muscle actin (ASMA) and have been found to play a major role in tissue contraction during wound healing and organ fibrosis. We therefore studied the expression of myofibroblasts in human primary knee arthrofibrosis tissue.Materials and methodsTissue samples were taken from the infrapatellar fat pad and intercondylar region of nine patients who underwent revision surgery due to arthrofibrosis after anterior cruciate ligament (ACL) reconstruction (study group). Control tissue was taken from five patients who underwent primary ACL reconstruction (control group I) and from eight patients, who underwent second-look arthroscopy after primary ACL reconstruction (control group II). ASMA containing fibroblasts were immunostained with a monoclonal antibody. Histomorphometry was performed for total cell amount, ASMA containing fibroblasts, and vessel cross-sections.ResultsThe arthrofibrosis group showed a tenfold higher amount of ASMA containing myofibroblasts (23.4% vs. 2.3%) than in control group I. There was a significantly higher total cell count and lower vessel density than in control group I. Control group II showed an upregulation of myofibroblasts almost five times that in control group I; nevertheless there was no evidence of scar formation or tissue fibrosis.ConclusionsMyofibroblasts are responsible for scar tissue contraction during wound healing. In arthrofibrosis tissue fibroblast contraction may be involved in tissue fibrosis and contraction with consecutive loss of motion. We found that myofibroblasts are upregulated in arthrofibrosis tissue. ACL reconstruction itself caused an up regulation of myofibroblast content. Nevertheless these patients did not show any clinical or histological signs of arthrofibrosis. Thus it is reasonable to assume that the ratio of myofibroblasts and total cell amount in connective tissue are responsible for the onset of arthrofibrosis. Address the expression of this highly differentiated cell type may therefore present a target for future therapeutic interventions.

Arthrofibrosis is the result of a T cell mediated immune response

Abstract. It is thought that an excessive fibrotic healing response with diffuse intra-articular scarring leads to arthrofibrosis after trauma and surgery around joints. To clarify the specific cellular mechanism of arthrofibrosis during arthrolysis we took fibrotic tissue samples from 18 patients at varying periods after knee trauma or surgery. Sections were stained with hematoxylin and eosin to study the overall histopathological changes. Major histocompatibility complex (MHC) class II expressing cells as well as CD3, CD4, CD25, CD28, CD68, CD80, and CD83 positive cells were localized immunohistologically. The results demonstrated synovial hyperplasia with fibrotic enlargement of the subintima and infiltration of inflammatory cells. The number of MHC class II expressing cells was increased. Mainly, intimal macrophages and dendritic cells showed positive immunostaining for MHC class II antigens. In the subintima moderate infiltration of T cells including activated T cells (CD25), CD4+ T helper (Th) cells and Th1 and Th2 subsets was detected. There was a slight polarization of the Th1/Th2 balance towards Th1 differentiation. Positive immunostaining for CD80/CD28 indicated the costimulatory signal for T cell activation and clonal expansion. These findings strongly support an immune response as the cause of capsulitis leading to formation of diffuse scar tissue within the knee joint. Based on our immunohistological study we conclude that a T cell mediated immune response plays a crucial role in the mechanism of arthrofibrosis.

Bone marrow stromal cells in a liquid fibrin matrix improve the healing process of patellar tendon window defects.

Following injury, ligaments and tendons do not regain their normal biological and biomechanical status. This study analyzed whether an injection of human bone marrow stromal cells (BMSC) or human fibroblast in a liquid fibrin matrix influences the histological results, ultrastructural morphology, mRNA expression of essential extracellular matrix proteins, and material properties of the healing tissue. Standardized full-thickness, full-length defects of the central portion of patellar tendons were created in 96 immunodeficient rats, and filled with human BMSC in a fibrin matrix (BMSC group), human fibroblasts in a fibrin matrix (fibroblast group), or fibrin matrix only (matrix group), or left untreated (defect group). Histological sections revealed more mature tissue formation with more regular patterns of cell distribution in the BMSC group, without signs of ectopic tissue formation into bone or cartilage. Mean collagen fibril diameter and relative area covered by collagen fibrils were significantly higher at 10 and 20 days postoperatively in the BMSC group compared to the defect and matrix groups, and comparable to normal tendon tissue. Further, collagen I mRNA expression, collagen I/collagen III mRNA ratio, and Youngs modulus were significantly increased at 20 days postoperatively in comparison to the defect and matrix groups. In the fibroblast group, only mean collagen fibril diameter was significantly higher compared to the defect group, whereas the other biological and biomechanical parameters were not significantly improved. This study reveals that an injection of BMSC in a liquid fibrin matrix stimulates histological, ultrastructural, molecular biologic, and biomechanical parameters of patellar tendon healing, whereas injection of fibroblasts in fibrin matrix had only minor effects on the stimulation of tendon healing.

Immunohistochemical localization of collagen VI in arthrofibrosis

Abstract Arthrofibrosis is a disabling complication after knee trauma and surgery. Clinically, it is characterized by pain and joint stiffness due to massive connective tissue proliferation. In similar pathological conditions with fibrotic transformation such as lung fibrosis or superficial fibromatoses, an increased expression of collagen type VI has been reported. Collagen VI, which forms a filamentous network, is thought to serve as an anchoring element between collagen I/III fibrils and basement membranes and as a cell binding structure. Collagen VI may also play a contributing role in the pathogenesis of arthrofibrosis. The aim of the present study was therefore to demonstrate the localization and distribution of type VI collagen in arthrofibrotic tissue. Tissue samples from the infrapatellar fat pad and intercondylar synovia of 13 patients suffering from arthrofibrosis were taken at surgery. The expression of type VI collagen was studied immunohistochemically using an immunoperoxidase method for light microscopic visualization. Histologic analysis showed a synovial hyperplasia with inflammatory cell infiltration and vascular proliferation. Compared with normal synovial tissue, type VI collagen was widely distributed as a network subsynovially and around the capillary walls. The results of the present study suggest that dysregulation of collagen VI synthesis could be an important contributing factor in the complex mechanisms of disordered matrix protein deposition leading to arthrofibrosis.