Guoguang Yang

Cyclic mechanical stretching of human tendon fibroblasts increases the production of prostaglandin E2 and levels of cyclooxygenase expression: a novel in vitro model study.

Prostaglandin E 2 (PGE 2 ) is a known inflammatory mediator of tendinitis, for which mechanical loading on tendons is believed to be one of the most prominent causation factors. Previous in vitro studies have shown that cyclic mechanical stretching of cells can cause changes in cell morphology and alteration of both DNA and protein syntheses. In our study, a novel system was used whereby tendon fibroblasts are cultured on microgrooved silicone surfaces and are subjected to cyclic uniaxial stretching along their long axes to mimic in vivo conditions. Using this unique model system, the cell shape and alignment can be controlled. Further, this study was designed to test the hypotheses that PGE 2 production increases in a stretching magnitude-dependent manner and that cyclooxygenase (COX) is responsible for the increased PGE 2 production in tendon fibroblasts. Human patellar tendon fibroblasts were cultured on the microgrooved silicone membranes and cyclically stretched at 4%, 8%, or 12% of nominal dish length for 24 hr. PGE 2 production was found to be increased 1.7-fold at 8% cyclic stretching and 2.2-fold at 12% cyclic stretching compared with nonstretched controls. In addition, human tendon fibroblasts had increased expression of both COX-1 and COX-2 for all three applied stretching magnitudes, with the exception of COX-1 at 4% cyclic stretching. Also, cellular PGE 2 production, after 8% cyclic stretching, was significantly decreased with the addition of indomethacin (25 w M), a COX competitive inhibitor, compared with stretched cells without indomethacin treatment. These findings suggest that the increase in PGE 2 production by the human tendon fibroblasts is stretching magnitude-dependent, and that the increase in COX expression contributes to the increased production of PGE 2 after cyclic stretching. As PGE 2 is a known inflammatory mediator of tendinitis, the contribution of COX-1 and COX-2 to PGE 2 production and their roles in tendon inflammation are clearly indicated.

Inflammatory response of human tendon fibroblasts to cyclic mechanical stretching.

Background The cellular and molecular mechanisms for the development of tendinopathy are not clear, but inflammatory mediators produced by tendon fibroblasts in response to repetitive mechanical loading may be an important factor. Hypotheses (1) Cyclic stretching of tendon fibroblasts affects the production of leukotriene B4 and the expression of 5-lipoxygenase; and (2) the production level of leukotriene B4 is inversely related to that of prostaglandin E2. Study Design Controlled laboratory study. Methods Human patellar tendon fibroblasts were uniaxially stretched in the presence of indomethacin (25 μM) or MK-886 (10 μM). After stretching for 4 hours, followed by 4 hours rest, levels of prostaglandin E2, leukotriene B4, and expression of 5-lipoxygenase were measured. Results Stretched tendon fibroblasts increased the levels of leukotriene B4 but did not appreciably change the expression of 5-lipoxygenase. Indomethacin decreased the cellular production of prostaglandin E2 but caused increased leukotriene B4 levels. MK-886 caused decreased production of leukotriene B4 but increased production of prostaglandin E2. Conclusions Cyclic stretching of human tendon fibroblasts increases the production of prostaglandin E2 and leukotriene B4. Blocking prostaglandin E2 production leads to increased leukotriene B4 levels and vice versa. Clinical Relevance The use of nonsteroidal anti-inflammatory drugs for the treatment of tendon inflammation might increase the levels of leukotriene B4 within the tendon, potentially contributing to the development of tendinopathy.

Synthesis, Biodegradability, and Biocompatibility of Lysine Diisocyanate–Glucose Polymers

The success of a tissue-engineering application depends on the use of suitable biomaterials that degrade in a timely manner and induce the least immunogenicity in the host. With this purpose in mind, we have attempted to synthesize a novel nontoxic biodegradable lysine diisocyanate (LDI)- and glucose-based polymer via polymerization of highly purified LDI with glucose and its subsequent hydration to form a spongy matrix. The LDI-glucose polymer was degradable in aqueous solutions at 37, 22, and 4 degrees C, and yielded lysine and glucose as breakdown products. The degradation products of the LDI-glucose polymer did not significantly affect the pH of the solution. The physical properties of the polymer were found to be adequate for supporting cell growth in vitro, as evidenced by the fact that rabbit bone marrow stromal cells (BMSCs) attached to the polymer matrix, remained viable on its surface, and formed multilayered confluent cultures with retention of their phenotype over a period of 2 to 4 weeks. These observations suggest that the LDI-glucose polymer and its degradation products were nontoxic in vitro. Further examination in vivo over 8 weeks revealed that subcutaneous implantation of hydrated matrix degraded in vivo three times faster than in vitro. The implanted polymer was not immunogenic and did not induce antibody responses in the host. Histological analysis of the implanted polymer showed that LDI-glucose polymer induced a minimal foreign body reaction, with formation of a capsule around the degrading polymer. The results suggest that biodegradable peptide-based polymers can be synthesized, and may potentially find their way into biomedical applications because of their biodegradability and biocompatibility.

Controlling Cell Responses to Cyclic Mechanical Stretching

In most cell culture studies, cells are grown on smooth culture surfaces. Using microfabrication technology, we have developed microgrooved silicone surfaces to grow cells and subject them to repetitive mechanical stretching. When human patellar tendon fibroblasts were plated on these microgrooved surfaces, the cells had an elongated shape and underwent cyclic uniaxial stretching parallel to their long axes, all of which closely mimic conditions of tendon fibroblasts in vivo. Also, when fibroblasts were grown on microgrooves oriented at 45 and 90 degrees with respect to stretching direction, they did not change alignment or shape under cyclic mechanical stretching. Furthermore, compared to nonstretched cells, 8% cyclic stretching of tendon fibroblasts oriented at 0 (i.e., parallel to stretching direction), 45, and 90 degrees was found to increase α-SMA protein expression level by 46, 31, and 14%, respectively. In addition, 8% cyclic stretching tendon fibroblasts for 4 and 8 h oriented parallel to stretching direction increased α-SMA protein expression level by 25 and 57%, respectively. Thus, the results of this study showed that α-SMA protein expression levels of tendon fibroblasts depend on cell orientation with respect to stretching direction and stretching duration. We suggest that microgrooved silicone substrates can be used to study biological responses of tendon or ligament fibroblasts to repetitive mechanical stretching conditions in a more controlled manner.

The Effects of Multiple Freeze–Thaw Cycles on the Biomechanical Properties of the Human Bone-Patellar Tendon-Bone Allograft

Soft tissue allografts, such as the bone‐patellar tendon‐bone (BPTB) graft, have been frequently used for anterior cruciate ligament (ACL) reconstruction. As allografts are subjected to freezing and thawing for multiple cycles, the objective of this study was to measure the changes of the biomechanical properties of the human BPTB allograft after 4 and 8 freeze–thaw cycles in comparison to a single freeze–thaw cycle. Three BPTB specimens were procured from 21 human donors and divided into three groups: 1, 4, or 8 freeze–thaw cycles. Each freeze–thaw cycle consisted of freezing at −20 ± 10°C for more than 6 h and thawing at 22 ± 3°C for at least 6 h. Tensile testing of the BPTB specimens consisted of loading between 50 N and 250 N for 100 cycles and then loading to failure. Cyclic loading revealed a similar amount of creep (∼0.5 mm) among the three freeze–thaw cycles groups (p = 0.38). The stiffness of the BPTB graft for the 1, 4, and 8 freeze–thaw cycle groups were 244 ± 42 N/mm, 235 ± 39 N/mm, and 231 ± 40 N/mm, respectively (p = 0.43). Similar findings were obtained for the ultimate load of the BPTB graft (p = 0.14) and the tangent modulus of the PT substance (p = 0.41). The results of this study suggest that there would be little measurable effect on the structural properties of the BPTB graft or mechanical properties of the PT tissue substance following 8 freeze–thaw cycles. These BPTB allografts could potentially be re‐frozen without a loss in their biomechanical properties, given appropriate storage and care.

Repetitively stretched tendon fibroblasts produce inflammatory mediators

We studied the expression of cytosolic phospholipase-A2 and activity of secretory phospholipase-A2 by human patellar tendon fibroblasts subjected to cyclic mechanical stretching. The effect of different stretching frequencies on the production of prostaglandin-E2 and expression of cyclooxygenase enzyme were also examined. An in vitro system that can control alignment, shape, and mechanical loading conditions of tendon fibroblasts was used for this study. Cyclic stretching of fibroblasts increased the expression level of cytosolic phospholipase-A2 by 88% and activity level of secretory phospholipase-A2 by 190%, compared with those of nonstretched fibroblasts. Cyclic stretching of tendon fibroblasts at 0.1 Hz and 1.0 Hz also increased prostaglandin-E2 production by 40% and 69%, respectively. Furthermore, cyclooxygenase-1 and cyclooxygenase-2 expression levels were increased in a stretching frequency-dependent manner, but cyclooxygenase-2 expression was increased more than that of cyclooxygenase-1. Because cytosolic phospholipase-A2 and secretory phospholipase-A2 are involved in the production of prostaglandin-E2 and other inflammatory mediators, this study suggests that regulation of phospholipase-A2 expression level may be an alternative approach to control in vivo tendon inflammation. The results of this study also may explain in part why activities that involve repetitive motion and high frequency loading of tendons are more likely to result in tendon inflammation.

Effect of Angelica sinensis on the proliferation of human bone cells.

BACKGROUND Angelica sinensis, an herbal medicine known for its effect to purify blood quality and improve circulation, frequently appears as the main ingredient in prescriptions for bone injuries. Currently, how pharmacologically it contributes to the reformation of bone is unclear. METHODS The effect of the aqueous extract of Angelica sinensis on bone cells was investigated in vitro for the first time. The human osteoprecursor cells (OPC-1) were incubated in the medium with different concentrations of the aqueous extract of Angelica sinensis and the cell proliferation was studied. RESULTS When the concentration of Angelica sinensis aqueous extract was <125 microg/ml, the proliferation of OPC-1 was enhanced. However, the proliferation of OPC-1 was inhibited by Angelica sinensis extract with the concentrations >250 microg/ml. Under most treatments, the cells presented very pale expression for cyclooxygenase-2 (Cox 2) protein; slightly intensified band showed at the highest Angelica sinensis concentration, 1.0 mg/ml during the course of culture. CONCLUSION The aqueous extract of Angelica sinensis was found to directly stimulate the proliferation, alkaline phosphatase (ALP) activity, protein secretion and particularly type I collagen synthesis of OPC-1 at dose-dependent manner.

Alpha1,3-galactosyltransferase knockout does not alter the properties of porcine extracellular matrix bioscaffolds

Extracellular matrix (ECM) bioscaffolds, such as porcine small intestine submucosa (SIS) and urinary bladder matrix (UBM), have been successfully used to improve soft tissue healing. Yet they contain plenty of galactose α1,3 galactose (αGal) epitopes, which cause rejection responses in pig organ transplantation to human. Recently, ECM bioscaffolds derived from genetically modified pigs that are αGal-deficient (αGal(-)) have become available. To ensure that the ECM bioscaffolds from these pigs can be used as alternatives, we examined their morphological, bioactive and biomechanical properties and compared them with those from the wild-type pigs (n=5 per group). Morphologically, the αGal(-) ECMs were found to be similar to the wild-type ECMs in gross observation and matrix appearance with hematoxylin and eosin staining. Growth factors commonly known to be present in ECM bioscaffolds, including FGF-2, TGF-β1, VEGF, IGF-1 and PDGF-BB, also showed no significant differences in terms of quantity (p>0.05) and distribution in tissue from the results of enzyme-linked immunosorbent assay, Western blot analysis and immunohistochemistry. Furthermore, a bromodeoxyuridine cell proliferation assay confirmed the bioactivity of the extracts from the αGal(-) bioscaffolds to be similar to the wild-type bioscaffolds. Under uniaxial tensile testing, no significant differences were found between the αGal(-) and wild-type bioscaffolds in terms of their viscoelastic and mechanical properties (p>0.05). These multidisciplinary results suggest that genetic modification to eliminate the αGal epitopes in the ECM bioscaffolds had not altered the properties of these ECM bioscaffolds and, as such, they should retain their performance in tissue engineering in humans.

Positive effects of an extracellular matrix hydrogel on rat anterior cruciate ligament fibroblast proliferation and collagen mRNA expression

Summary Background/Objective We have previously shown that an extracellular matrix (ECM) bioscaffold derived from porcine small intestine submucosa (SIS) enhanced the healing of a gap injury of the medial collateral ligament as well as the central third defect of the patellar tendon. With the addition of a hydrogel form of SIS, we found that a transected goat anterior cruciate ligament (ACL) could also be healed. The result begs the research question of whether SIS hydrogel has positive effects on ACL fibroblasts (ACLFs) and thus facilitates ACL healing. Methods In the study, ECM-SIS hydrogel was fabricated from the digestion of decellularised and sterilised sheets of SIS derived from αGal-deficient (GalSafe) pigs. As a comparison, a pure collagen hydrogel was also fabricated from commercial collagen type I solution. The morphometrics of hydrogels was assessed with scanning electron microscopy. The ECM-SIS and collagen hydrogels had similar fibre diameters (0.105 ± 0.010 μm vs. 0.114 ± 0.004 μm), fibre orientation (0.51 ± 0.02 vs. 0.52 ± 0.02), and pore size (0.092 ± 0.012 μm vs. 0.087 ± 0.008 μm). The preservation of bioactive properties of SIS hydrogel was assessed by detecting bioactive molecules sensitive to processing and enzyme digestion, such as growth factors fibroblast growth factor-2 (FGF-2) and transforming growth factor-beta 1 (TGF-β1), with enzyme-linked immunosorbent assay. ACLFs were isolated and expanded in culture from explants of rat ACLs (n = 3). The cells were then seeded on the hydrogels and cultured with 0%, 1%, and 10% foetal bovine serum (FBS) for 3 days and 7 days. Cell attachment was observed using a light microscope and scanning electron microscopy, whereas cell proliferation and matrix production (collagen types I and III) were examined with bromodeoxyuridine assays and reverse transcription-polymerase chain reaction, respectively. Results The results showed that FGF-2 and TGF-β1 in the SIS hydrogel were preserved by 50% (65.9 ± 26.1 ng/g dry SIS) and 90% (4.4 ± 0.6 ng/g dry SIS) relative to their contents in ECM-SIS sheets, respectively. At Day 3 of culture, ACLFs on the SIS hydrogel were found to proliferate 39%, 31%, and 22% more than those on the pure collagen hydrogel at 0%, 1%, and 10% FBS, respectively (p < 0.05). Collagen type I mRNA expression was increased by 150%, 207%, and 100%, respectively, compared to collagen hydrogel (p < 0.05), whereas collagen type III mRNA expression was increased by 123% and 132% at 0% and 1% FBS, respectively (all p < 0.05) but not at 10% FBS. By Day 7, collagen type I mRNA expression was still elevated by 137% and 100% compared to collagen hydrogel at 1% and 10% FBS, respectively (p < 0.05). Yet, collagen type III mRNA levels were not significantly different between the two groups at any FBS concentrations. Conclusion Our data showed that the ECM-SIS hydrogel not only supported the growth of ACLFs, but also promoted their proliferation and matrix production relative to a pure collagen hydrogel. As such, ECM-SIS hydrogel has potential therapeutic value to facilitate ACL healing at the early stage after injury.

Characterization of αGal(−) ECM Bioscaffolds: An Indication for Application in Tissue Engineering

Extracellular matrix (ECM) bioscaffolds derived from animal sources, such as porcine small intestine submucosa (SIS) and urinary bladder matrix (UBM), have a successful history for application in clinical and/or experimental settings [1]. In our research center, we have demonstrated that the porcine SIS bioscaffolds could improve the morphological, biochemical, and biomechanical properties of healing ligaments and tendons in different animal models [2, 3].Copyright