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Eur J Biochem. 2000 Dec;267(23):6943-50.
Characterization of a rainbow trout matrix metalloproteinase capable of degrading type I collagen.
Saito M, Sato K, Kunisaki N, Kimura S.
Laboratory of Food Science, Kagawa Nutrition University, Komagome, Toshima, Tokyo, Japan. msaitiyo.ac.jp
Matrix metalloproteinases (MMPs) are widely distributed in vertebrate tissues and form a large family consisting of at least four distinct subfamilies. Higher vertebrate MMP-13 is well-known as collagenase-3, which represents the third member of a collagenase subfamily. In this study, we cloned cDNA coding for a unique fish homologue of human MMP-13 from a rainbow trout fibroblast cDNA library. The cDNA was 2.1 kb long and contained an open reading frame encoding a protein of 475 amino acids. The catalytic domain of the protein was 66% identical to the human counterpart with the greatest degree of identity occurring in the zinc binding site. In addition, it possessed three amino-acid residues (Tyr122, Asp233 and Gly235) characteristic of the collagenase subfamily, together with a six residue insertion which did not occur in the collagenase subfamily. Then the isolated cDNA was expressed in Escherichia coli and the recombinant protein was found to degrade gelatin and skin type I collagen. It is worth noting that rainbow trout type I collagen was more susceptible to proteolysis with the recombinant protein when compared with the calf one. It appeared that the recombinant protein also cleaved the nonhelical regions of rainbow trout muscle type V collagen. These results have revealed that the cDNA encodes a unique MMP-13 of rainbow trout. This is the first report of cDNA coding for fish MMP capable of degrading type I collagen.
online pharmacy ref. source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11082208&dopt=Abstract
Biomaterials. 2003 Feb;24(4):587-96.
In vitro behaviour of bone marrow-derived mesenchymal cells cultured on fluorohydroxyapatite-coated substrata with different roughness.
Campoccia D, Arciola CR, Cervellati M, Maltarello MC, Montanaro L.
Research Laboratory on Biocompatibility of Implant Materials, Rizzoli Orthopaedic Institute, Via di Barbiano 1/10, 40136 Bologna, Italy.
Among ceramic materials, recent interest has been risen on fluorinated hydroxyapatites, which undergo slow in vivo degradation and offer a more stable interface for osseointegration and bone fixation. Apart from the information available on the chemistry, little is known of their biological properties and, more specifically, of their interaction with bone cells. The aim of the present study has been to investigate the behaviour and differentiation of human bone marrow-derived mesenchymal cells cultured on two substrata consisting of fluorohydroxyapatite (FHA)-coated titanium and characterised by different surface roughness. To this end, osteoprogenitor cells were seeded on the test materials and, once adhered, were induced to differentiate to osteoblastic cells. The cell behaviour on the different surfaces was monitored for a period of up to 2 weeks. The results obtained indicate that FHAs are cytocompatible materials, which allow the adhesion and growth of osteogenic cells. Mesenchymal cells promptly adhered, covering the surface of the test materials, and subsequently expressed some typical markers of osteoblasts. No significant difference in alkaline phosphatase specific activity was observed when comparing the two test material surfaces to plastic control. At 7 days the number of adhered cells and the presence in the medium of C-terminal propeptide of type I collagen appeared lower or slightly lower for cultures on FHA substrata than on plastic control. These differences tended to subside with time.
online pharmacy ref. source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12437953&dopt=Abstract
Circulation. 2000 Nov 7;102(19 Suppl 3):III22-9.
Early in vivo experience with tissue-engineered trileaflet heart valves.
Sodian R, Hoerstrup SP, Sperling JS, Daebritz S, Martin DP, Moran AM, Kim BS, Schoen FJ, Vacanti JP, Mayer JE Jr.
Department of Cardiac Research, Children's Hospital Boston, Harvard Medical School, Boston, MA 02115, USA.
BACKGROUND: Tissue engineering is a new approach in which techniques are being developed to transplant autologous cells onto biodegradable scaffolds to ultimately form new functional autologous tissue. Workers at our laboratory have focused on tissue engineering of heart valves. The present study was designed to evaluate the implantation of a whole trileaflet tissue-engineered heart valve in the pulmonary position in a lamb model. METHODS AND RESULTS: We constructed a biodegradable and biocompatible trileaflet heart valve scaffold that was fabricated from a porous polyhydroxyalkanoate (pore size 180 to 240 microm; Tepha Inc). Vascular cells were harvested from ovine carotid arteries, expanded in vitro, and seeded onto our heart valve scaffold. With the use of cardiopulmonary bypass, the native pulmonary leaflets were resected, and 2-cm segments of pulmonary artery were replaced by autologous cell-seeded heart valve constructs (n=4). One animal received an acellular valved conduit. No animal received any anticoagulation therapy. Animals were killed at 1, 5, 13, and 17 weeks. Explanted valves were examined histologically with scanning electron microscopy, biochemically, and biomechanically. All animals survived the procedure. The valves showed minimal regurgitation, and valve gradients were <20 mm Hg on echocardiography. The maximum gradient was 10 mm Hg with direct pressures. Macroscopically, the tissue-engineered constructs were covered with tissue, and there was no thrombus formation on any of the specimens. Scanning electron microscopy showed smooth flow surfaces during the follow-up period. Histological examination demonstrated laminated fibrous tissue with predominant glycosaminoglycans as extracellular matrix. 4-Hydroxyproline assays demonstrated an increase in collagen content as a percentage of native pulmonary artery (1 week 45.8%, 17 weeks 116%). DNA assays showed a comparable number of cells in all explanted samples. There was no tissue formation in the acellular control. CONCLUSIONS: Tissue-engineered heart valve scaffolds fabricated from polyhydroxyalkanoates can be used for implantation in the pulmonary position with an appropriate function for 120 days in lambs.
online pharmacy ref. source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11082357&dopt=Abstract
Circulation. 2000 Nov 7;102(19 Suppl 3):III44-9.
Functional living trileaflet heart valves grown in vitro.
Hoerstrup SP, Sodian R, Daebritz S, Wang J, Bacha EA, Martin DP, Moran AM, Guleserian KJ, Sperling JS, Kaushal S, Vacanti JP, Schoen FJ, Mayer JE Jr.
Department of Cardiovascular Surgery, Children's Hospital Boston, MA, USA. simon_philipp.hoerstruhir.usz.ch
BACKGROUND: Previous tissue engineering approaches to create heart valves have been limited by the structural immaturity and mechanical properties of the valve constructs. This study used an in vitro pulse duplicator system to provide a biomimetic environment during tissue formation to yield more mature implantable heart valves derived from autologous tissue. METHODS AND RESULTS: Trileaflet heart valves were fabricated from novel bioabsorbable polymers and sequentially seeded with autologous ovine myofibroblasts and endothelial cells. The constructs were grown for 14 days in a pulse duplicator in vitro system under gradually increasing flow and pressure conditions. By use of cardiopulmonary bypass, the native pulmonary leaflets were resected, and the valve constructs were implanted into 6 lambs (weight 19+/-2.8 kg). All animals had uneventful postoperative courses, and the valves were explanted at 1 day and at 4, 6, 8, 16, and 20 weeks. Echocardiography demonstrated mobile functioning leaflets without stenosis, thrombus, or aneurysm up to 20 weeks. Histology (16 and 20 weeks) showed uniform layered cuspal tissue with endothelium. Environmental scanning electron microscopy revealed a confluent smooth valvular surface. Mechanical properties were comparable to those of native tissue at 20 weeks. Complete degradation of the polymers occurred by 8 weeks. Extracellular matrix content (collagen, glycosaminoglycans, and elastin) and DNA content increased to levels of native tissue and higher at 20 weeks. CONCLUSIONS: This study demonstrates in vitro generation of implantable complete living heart valves based on a biomimetic flow culture system. These autologous tissue-engineered valves functioned up to 5 months and resembled normal heart valves in microstructure, mechanical properties, and extracellular matrix formation.
online pharmacy ref. source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11082361&dopt=Abstract
Circulation. 2000 Nov 7;102(19 Suppl 3):III50-5.
Tissue engineering of pulmonary heart valves on allogenic acellular matrix conduits: in vivo restoration of valve tissue.
Steinhoff G, Stock U, Karim N, Mertsching H, Timke A, Meliss RR, Pethig K, Haverich A, Bader A.
Leibniz Research Laboratories for Biotechnology and Artificial Organs Department of Thoracic and Cardiovascular Surgery, Medical School Hannover, Hannover, Germany.
BACKGROUND: Tissue engineering using in vitro-cultivated autologous vascular wall cells is a new approach to biological heart valve replacement. In the present study, we analyzed a new concept to process allogenic acellular matrix scaffolds of pulmonary heart valves after in vitro seeding with the use of autologous cells in a sheep model. METHODS AND RESULTS: Allogenic heart valve conduits were acellularized by a 48-hour trypsin/EDTA incubation to extract endothelial cells and myofibroblasts. The acellularization procedure resulted in an almost complete removal of cells. After that procedure, a static reseeding of the upper surface of the valve was performed sequentially with autologous myofibroblasts for 6 days and endothelial cells for 2 days, resulting in a patchy cellular restitution on the valve surface. The in vivo function was tested in a sheep model of orthotopic pulmonary valve conduit transplantation. Three of 4 unseeded control valves and 5 of 6 tissue-engineered valves showed normal function up to 3 months. Unseeded allogenic acellular control valves showed partial degeneration (2 of 4 valves) and no interstitial valve tissue reconstitution. Tissue-engineered valves showed complete histological restitution of valve tissue and confluent endothelial surface coverage in all cases. Immunohistological analysis revealed cellular reconstitution of endothelial cells (von Willebrand factor), myofibroblasts (alpha-actin), and matrix synthesis (procollagen I). There were histological signs of inflammatory reactions to subvalvar muscle leading to calcifications, but these were not found in valve and pulmonary artery tissue. CONCLUSIONS: The in vitro tissue-engineering approach using acellular matrix conduits leads to the in vivo reconstitution of viable heart valve tissue.
online pharmacy ref. source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11082362&dopt=Abstract
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