Influence of cyclical mechanical loading on osteogenic markers in an osteoblast-fibroblast co-culture in vitro: tendon-to-bone interface in anterior cruciate ligament reconstruction.

PURPOSE: We aimed to evaluate the influence of cyclical mechanical loading on osteoblasts and fibroblasts, and co-cultures of both in vitro, simulating the conditions of the tendon-to-bone interface in anterior cruciate ligament reconstruction. METHODS: Osteoblast-like cells (OBL) and tendon-derived rodent fibroblasts (TDF) were cultured alone or in co-culture to simulate the tendon-to-bone interface. Cyclical loading was applied for one hour twice a day for three days, with a frequency of 1 Hz and 3 % strain. Alkaline phosphatase (AP), osteocalcin (OC), collagen type 1 (COL1A1), and bone morphogenetic protein 2 (BMP-2) gene expression and protein deposition were detected by real-time polymerase chain reaction (qPCR) and immunocytochemical analysis. RESULTS: Mechanical loading significantly decreased AP, OC, and COL1A1 gene expression in both OBL and TDF, compared to non-loaded culture. However, mechanical load increased gene expression of the same marker genes including BMP-2 during co-culture. Immunocytochemistry demonstrated increased deposition of corresponding proteins in the same range, independent of culture conditions. Higher depositions of BMP-2 were shown under loading conditions for osteoblast and TDF monocultures. Prolongation of mechanical loading resulted in cell detachment and spheroid formation. CONCLUSION: Cyclical mechanical loading caused downregulation of genes involved in osteointegration and osteoinduction, such as OC, ALP, and COL1A1 in monocultures of osteoblasts and fibroblasts; co-cultures lacked this phenomenon. Immunocytochemistry and qPCR analysis showed slight upregulations of marker genes and corresponding proteins. This might be due to the potential stabilising effects of osteoblast-fibroblast cross talk in the co-culture environment, simulating fibrocartilage formation at the tendon-to-bone interface.

Expression of matrix metalloproteinases 1, 3, and 9 in differing extents of tendon retraction in the torn rotator cuff.

PURPOSE: Differing extents of tendon retraction are found in full-thickness rotator cuff tears. The pathophysiologic context of tendon degeneration and the extent of tendon retraction are unclear. Tendon integrity depends on the extracellular matrix, which is regulated by matrix metalloproteinases (MMP). It is unknown which enzymes play a role in tendon degeneration. The hypotheses are that (1) the expression of MMPs 1, 3, and 9 is altered in the torn rotator cuff when compared with healthy tendon samples; and (2) that there is a relationship between MMP expression and the extent of tendon retraction in the torn cuff. METHODS: Rotator cuff tendon samples of 33 patients with full-thickness rotator cuff tears (Bateman grade III) were harvested during reconstructive surgery. Samples were dehydrated and paraffin-embedded. Immunohistologic determination of MMP 1, 3, and 9 expression was performed by staining sample slices with MMP antibody. The extent of tendon retraction was determined intraoperatively according to Patte's classification and patients were assigned to 4 groups (control group, and by tendon retraction grade Patte I-III). The control group consisted of six healthy tendon samples. RESULTS: Expression of MMPs 1 and 9 was significantly higher in torn cuff samples than in healthy tendons whereas MMP 3 expression was significantly decreased (P < 0.05). MMP 9 expression significantly increased with rising extent of tendon retraction in the torn cuff (P < 0.05). No significant association was found between expression of MMPs 1 and 3 and the rising extent of tendon retraction by Patte's classification. CONCLUSION: Elevated expression of MMPs 1 and 9 as well as decreased MMP 3 expression can be detected in torn rotator cuff tendon tissue. There is a significant association between the extent of tendon retraction and MMP 9 expression. The results of this study give evidence that early surgical treatment of small and partial-thickness rotator cuff tears is required.

Expression of Estrogen Receptor Alpha and Evaluation of Histological Degeneration Scores in Fibroblasts of Hypertrophied Ligamentum Flavum: A Qualitative Study.

The most common spinal disorder in elderly is lumbar spinal stenosis (LSS), resulting partly from ligamentum flavum (LF) hypertrophy. Its pathophysiology is not completely understood. The present study wants to elucidate the role of estrogen receptor α (ER α) in fibroblasts of hypertrophied LF. LF samples of 38 patients with LSS were obtained during spinal decompression. Twelve LF samples from patients with disk herniation served as controls. Hematoxylin & Eosin (H&E) and Elastica stains and immunohistochemistry for ER α were performed. The proportions of fibrosis, loss and/or degeneration of elastic fibers and proliferation of collagen fibers were assessed according to the scores of Sairyo and Okuda. Group differences in the ER α and Sairyo and Okuda scores between patients and controls, male and female sex and absence and presence of additional orthopedic diagnoses were assessed with the Mann-Whitney U test. There was a tendency towards higher expression of ER α in LF fibroblasts in the hypertrophy group (p = 0.065). The Sairyo and Okuda scores were more severe for the hypertrophy group but, in general, not statistically relevant. There was no statistically relevant correlation between the expression of ER α and sex (p = 0.326). ER α expression was higher in patients with osteochondrosis but not statistically significant (p = 0.113). In patients with scoliosis, ER α expression was significantly lower (p = 0.044). LF hypertrophy may be accompanied by a higher expression of ER α in fibroblasts. No difference in ER α expression was observed regarding sex. Further studies are needed to clarify the biological and clinical significance of these findings.

Influence of nanofibers on growth and gene expression of human tendon derived fibroblast.

BACKGROUND: Rotator cuff tears are a common and frequent lesion especially in older patients. The mechanisms of tendon repair are not fully understood. Common therapy options for tendon repair include mini-open or arthroscopic surgery. The use of growth factors in experimental studies is mentioned in the literature. Nanofiber scaffolds, which provide several criteria for the healing process, might be a suitable therapy option for operative treatment. The aim of this study was to explore the effects of nanofiber scaffolds on human tendon derived fibroblasts (TDF's), as well as the gene expression and matrix deposition of these fibroblasts. METHODS: Nanofibers composed of PLLA and PLLA/Col-I were seeded with human tendon derived fibroblasts and cultivated over a period of 22 days under growth-inductive conditions, and analyzed during the course of culture, with respect to gene expression of different extra cellular matrix components such as collagens, bigylcan and decorin. Furthermore, we measured cell densities and proliferation by using fluorescence microscopy. RESULTS: PLLA nanofibers possessed a growth inhibitory effect on TDF's. Furthermore, no meaningful influence on the gene expression of collagen I, collagen III and decorin could be observed, while the expression of collagen X increased during the course of cultivation. On the other hand, PLLA/Col-I blend nanofibers had no negative influence on the growth of TDF's. Furthermore, blending PLLA nanofibers with collagen had a positive effect on the gene expression of collagen I, III, X and decorin. Here, gene expression indicated that focal adherence kinases might be involved. CONCLUSION: This study indicates that the use of nanofibers influence expression of genes associated with the extra cellular matrix formation. The composition of the nanofibers plays a critical role. While PLLA/Col-I blend nanofibers enhance the collagen I and III formation, their expression on PLLA nanofibers was more comparable to controls. However, irrespective of the chemical composition of the fibres, the collagen deposition was altered, an effect which might be associated with a decreased expression of biglycanes.

The relevance of long head biceps degeneration in the presence of rotator cuff tears.

BACKGROUND: Long head biceps (LHB) degeneration in combination with rotator cuff tears can be a source of chronic shoulder pain. LHB tenotomy is an approved surgical procedure for pain reduction and improvement of joint function, however, the pathophysiology of LHB degeneration is not fully understood. In the literature, neoangiogenesis in tendon tissue has previously been shown to be associated with tendon degeneration. Vascular Endothelial Growth Factor (VEGF) is an important inducer of neoangiogenesis. The hypotheses are first that an elevated VEGF expression and vessel density can be found in degenerated LHB tissue and second that there is a relation between VEGF expression, vessel density and the different types of rotator cuff tears. METHODS: LHB samples of 116 patients with degenerative rotator cuff tears were harvested during arthroscopic tenotomy. The samples were dehydrated and paraffin embedded. VEGF expression was determined using immunohistochemistry. Vessel density and vessel size were determined on Masson-Goldner stained tissue sections. On the basis of intraoperative findings, patients were assigned to 4 different groups (control group, partial thickness rotator cuff tear, full thickness rotator cuff tear and cuff arthropathy). Partial thickness rotator cuff tears were classified according to Ellman grade I-III, full thickness rotator cuff tears according to Bateman's classification (grade I-IV). The control group consisted of eight healthy tendon samples. RESULTS: VEGF expression in the LHB was significantly higher in the presence of rotator cuff tears than in healthy tendons (p < 0.05) whereas vessel density and vessel size were significantly higher in the LHB of patients with cuff arthropathy (p < 0.05). Furthermore, there was significantly higher VEGF expression in LHB samples from patients with articular-sided compared to bursal-sided partial thickness rotator cuff tears (p < 0.05). No significant dependence was found between VEGF expression, vessel size and vessel density in LHB of patients with full thickness rotator cuff tears and the extent of the cuff tear following Bateman's classification. CONCLUSION: Elevated VEGF expression can be detected in degenerated LHB tissue. The quantity of VEGF expression and vessels are related to the extent of LHB degeneration.

The association between retraction of the torn rotator cuff and increasing expression of hypoxia inducible factor 1α and vascular endothelial growth factor expression: an immunohistological study.

BACKGROUND: Differing levels of tendon retraction are found in full-thickness rotator cuff tears. The pathophysiology of tendon degeneration and retraction is unclear. Neoangiogenesis in tendon parenchyma indicates degeneration. Hypoxia inducible factor 1α (HIF) and vascular endothelial growth factor (VEGF) are important inducers of neoangiogenesis. Rotator cuff tendons rupture leads to fatty muscle infiltration (FI) and muscle atrophy (MA). The aim of this study is to clarify the relationship between HIF and VEGF expression, neoangiogenesis, FI, and MA in tendon retraction found in full-thickness rotator cuff tears. METHODS: Rotator cuff tendon samples of 33 patients with full-thickness medium-sized rotator cuff tears were harvested during reconstructive surgery. The samples were dehydrated and paraffin embedded. For immunohistological determination of VEGF and HIF expression, sample slices were strained with VEGF and HIF antibody dilution. Vessel density and vessel size were determined after Masson-Goldner staining of sample slices. The extent of tendon retraction was determined intraoperatively according to Patte's classification. Patients were assigned to 4 categories based upon Patte tendon retraction grade, including one control group. FI and MA were measured on standardized preoperative shoulder MRI. RESULTS: HIF and VEGF expression, FI, and MA were significantly higher in torn cuff samples compared with healthy tissue (p < 0.05). HIF and VEGF expression, and vessel density significantly increased with extent of tendon retraction (p < 0.05). A correlation between HIF/VEGF expression and FI and MA could be found (p < 0.05). There was no significant correlation between HIF/VEGF expression and neovascularity (p > 0.05) CONCLUSION: Tendon retraction in full-thickness medium-sized rotator cuff tears is characterized by neovascularity, increased VEGF/HIF expression, FI, and MA. VEGF expression and neovascularity may be effective monitoring tools to assess tendon degeneration.

Expression of matrix metalloproteinases 1, 3, and 9 in degenerated long head biceps tendon in the presence of rotator cuff tears: an immunohistological study.

BACKGROUND: Long head biceps (LHB) degeneration, in combination with rotator cuff tears, can be a source of chronic shoulder pain. LHB tenotomy reduces pain and improves joint function although the pathophysiological context is not well understood. Tendon integrity depends on the extracellular matrix (ECM), which is regulated by matrix metalloproteinases (MMP). It is unclear which of these enzymes contribute to LHB but we chose to study MMP 1, 3, and 9 and hypothesized that one or more of them may be altered in LHB, whether diagnosed preoperatively or intraoperatively. We compared expression of these MMPs in both LHB and healthy tendon samples. METHODS: LHB samples of 116 patients with degenerative rotator cuff tears were harvested during arthroscopic tenotomy. Patients were assigned to 4 groups (partial thickness tear, full thickness tear, cuff arthropathy, or control) based upon intraoperative findings. Partial and full thickness tears were graded according to Ellman and Bateman's classifications, respectively. MMP expression was determined by immunohistochemistry. RESULTS: MMP 1 and 9 expression was significantly higher in the presence of rotator cuff tears than in controls whereas MMP 3 expression was significantly decreased. MMP 1 and 9 expression was significantly higher in articular-sided than bursal-sided partial thickness tears. No significant association was found between MMP 1 and 9 expression and full thickness tears, and the extent of the cuff tear by Bateman's classification. CONCLUSION: Increased MMP 1 and 9 expression, and decreased MMP 3 expression are found in LHB degeneration. There is a significant association between the size and location of a rotator cuff tear and MMP expression.