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|Número de publicación
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|Número de solicitud
|Fecha de publicación
|23 Dic 1997
|Fecha de presentación
|20 Oct 1995
|Fecha de prioridad
|20 Oct 1995
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|Arnold S. Breitbart, Daniel A. Grande
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|North Shore University Hospital Research Corporation
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|Citas de patentes (10), Citada por (174), Clasificaciones (16), Eventos legales (7)
|Enlaces externos: USPTO, Cesión de USPTO, Espacenet
The present invention is generally in the area of methods for repair and reconstruction of bone.
Bone is built of a dense network of collagen protein fibers arranged in layers with crystals of calcium phosphate and calcium carbonate between the fibers. About 25% of the bone's weight is calcium. About four percent of the bone's volume, scattered evenly throughout it, are living cells called osteocytes. These are supplied with oxygen and nutrients through a network of very small blood vessels that extend throughout the bone. Defects in the bone are repaired by osteoclasts removing the damaged bone, then osteoblast cells laying down new bone. The osteoblasts repeatedly form layers, each consisting of a network of collagen fibers, which produce enzymes resulting in calcium and phosphorus deposition, until the defect is repaired.
Bone repair has been primarily achieved using bone cements, pins, screws and other devices for mechanical stabilization. Larger defects, however, arising from trauma or surgery, require replacement of the missing bone with a material that provides support and which can be immobilized, yet which is also biocompatible. A graft may be necessary when bone fails and does not repair itself in the normal amount of time or when bone loss occurs through fracture or tumor. Bone grafts must serve a dual function: to provide mechanical stability and to be a source of osteogenesis. Since skeletal injuries are repaired by the regeneration of bone rather than by the formation of scar tissue, grafting is a viable means of promoting healing of osseous defects, as reviewed by Friedlaender, G. E., "Current Concepts Review: Bone Grafts," Journal of Bone and Joint Surgery, 69A(5), 786-790 (1987). Osteoinduction and osteoconduction are two mechanisms by which a graft may stimulate the growth of new bone. In the former case, inductive signals of little-understood nature lead to the phenotypic conversion of connective tissue cells to bone cells. In the latter, the implant provides a scaffold for bony ingrowth. The bone remodeling cycle is a continuous event involving the resorption of pre-existing bone by osteoclasts and the formation of new bone by the work of osteoblasts. Normally, these two phases are synchronous and bone mass remains constant. However, the processes become uncoupled when bone defects heal and grafts are incorporated. Osteoclasts resorb the graft, a process which may take months. More porous grafts revascularize more quickly and graft resorption is more complete. After the graft has been resorbed, bone formation begins. Bone mass and mechanical strength return to near normal.
Present methods for the repair of bony defects include grafts of organic and synthetic construction. Three types of organic grafts are commonly used: autografts, allografts, and xenografts. An autograft is tissue transplanted from one site to another in the patient. The benefits of using the patient's tissue are that the graft will not evoke a strong immune response and that the material may or may not be vascularized, which allows for speedy incorporation. However, using an autograft requires a second surgery, which increases the risk of infection and introduces additional weakness at the harvest site. Further, bone available for grafting may be removed from a limited number of sites, for example, the fibula, ribs and iliac crest. An allograft is tissue taken from a different organism of the same species, and a xenograft from an organism of a different species. The latter types of tissue are readily available in larger quantities than autografts, but genetic differences between the donor and recipient may lead to rejection of the graft. Periosteal and perichonchondral grafting has also been attempted, as described by Ritsila, et al., Clin. Orthop. Related Res. 302, 259-265 (1994). Examples of synthetic materials which have been used include titanium and steel alloys, particularly those having a porous structure to allow ingrowth of cells to stabilize the implant, bone cements, alone or mixed with cells, sterilized bone, and polymeric or polymeric/hydroxyapatite implants. Bioerodible polymers have been used in people for thousands of years, with plain gut (collagen) sutures being used since 175 A.D.(12), as reported by Chu, In Biocompatible Polymers, Metals and Composites, ed. Szycher, M, p. 477 (Technomic Publishing Lakewood, N.J. 1983). The first synthetic biodegradable biopolymers, polylactic acid and polyglycolic acid, were suggested for in vivo use in U.S. Pat. No. 3,371,069 to Schmidt. All have advantages and disadvantages, yet none provides a perfect replacement for the missing bone.
Large defects are particularly difficult. One approach has been to seed fibrous biodegradable polymeric matrices with bone-forming cells, then overlay the matrix onto the defect. As the cells proliferate, and surrounding tissues grow into the defect, the matrix will degrade, leaving the new tissue. As described in U.S. Pat. No. 4,846,835 to Grande and U.S. Pat. No. 5,041,138 to Vacanti, et al., cartilage has been grown by seeding synthetic polymeric matrices with dissociated cells, which are then implanted to form new cartilage.
The first description of bone cells in culture was by Peck, et al., in Science 146, 1476 (1964). Since that time, many studies have focused on the maintenance of viable cell cultures of osteoblasts with full expression of phenotype, as discussed by Peck, et al., Endocrinology 92, 692 (1982). Other studies examining bone cell growth and activity regulation in vitro have identified various factors necessary for cell development, as reported by Beresfor, et al., Calcif. Tissue Int. 35, 637 (1983). Studies on osteoblast growth on supports outside of the traditional tissue culture environment have concentrated on studying the growth of these cells on mineral matrices which mimic the natural hydroxyapatite environment in vivo, as reported by Chueng and Haak, Biomaterials 10, 63 (1989). Hydroxyapatite (HA), Ca3 (PO4)2.Ca(OH)2, is a natural mineral structure that resembles the crystal lattice of bones. Studies on the growth of osteoblasts in culture on calcium phosphate ceramic surfaces demonstrated that osteoblasts, fibroblasts and chondrocytes attach to the ceramic material and form multicellular layers. Retention of phenotypic activity of osteoblasts was demonstrated through parathyroid hormone suppression of alkaline phosphatase activity, and cAMP increase as well as expression of Type I collagen.
The cell source is in some cases determinative of the usefulness of this method. It is clearly most desirable to use a patient's own cells to repair a defect, thereby avoiding problems with immune rejection or contamination. Sources of cells include growing and mature bone, cartilage, and mesenchymal stem cells. Chondro/osteoprogenitor cells, which are bipotent with the ability to differentiate into cartilage or bone, have been isolated from bone marrow (for example, as described by Owen, J. Cell Sci. Suppl. 10, 63-76 (1988) and in U.S. Pat. No. 5,226,914 to Caplan, et al.). These cells led Owen to postulate the existence of pluripotent mesenchymal stem cells, which were subsequently isolated from muscle (Pate, et al., Proc. 49th Ann. Sess. Forum Fundamental Surg. Problems 587-589 (Oct. 10-15, 1993)), heart (Dalton, et al., J. Cell Biol. 119, R202 (March 1993)), and granulation tissue (Lucas, et al., J. Cell Biochem. 122, R212 (March 1993)). Pluripotency is demonstrated using a non-specific inducer, dexamethasone (DMSO), which elicits differentiation of the stem cells into chondrocytes (cartilage), osteoblasts (bone), myotubes (muscle), adipocytes (fat), and connective tissue cells. There remains a need for a ready source of cells for use in repairing bone defects.
It is an object of the present invention to provide a method and materials for repair of bone defects, using the patient as the source of cells to thereby avoid immune rejection.
It is a further object of the present invention to provide a method and materials for repair of bone defects, which allows repair of large defects.
It is a further object of the present invention to provide a method and materials for repair of bone defects which ultimately results solely in formation of new bone, without problems relating to long term implantation of foreign materials which could elicit inflammation, bioincompatibility, and poor union with the patient's own bone.
Methods for repair of bone are disclosed where cells are obtained from periosteum, dissociated, then seeded into an appropriate matrix for repair of the defect. Periosteum consists of multipotent mesodermal cells. The cells are isolated, seeded onto a matrix for implantation, then cultured under conditions which induce the cells to form bone rather than other tissue types, especially cartilage. A variety of different matrix materials can be used to support the dissociated cells. In a preferred embodiment, the material is a biodegradable, biocompatible synthetic polymer such as fibrous polyglycolic acid. Hydrogel matrices can also be used.
Periosteum has been demonstrated to have cell populations that can be isolated in culture, forming both cartilage and bone. In the following example, periosteal cells cultured under osteogenic conditions were used to repair critical size cranial defects in a rabbit model. Periosteum was isolated from New Zealand White (NSW) rabbits, grown in culture, labeled with BrDU, and the cells were seeded into polyglycolic acid (PGA) matrices. Adult NZW rabbits were divided into control and experimental animals and a single 15 mm diameter, full-thickness calvarial defect was made in each rabbit. In the experimental animals, defects were repaired using PGA implants seeded with periosteal cells. In control animals, defects were repaired with untreated PGA implants.
FIG. 1 is a graph of the total glycosaminoglycan (GAG) in periosteal tissue constructs in vitro, micrograms GAG/mg dry weight over time (days), for dexamethasone:GAG/dry (squares) and dexamethasone (diamond).
FIG. 2 is a graph of the osteocalcin levels, osteocalcin (ng/ml) at day 7, day 14, and day 21, with dexamethasone (dots) and without dexamethasone (solid).
FIGS. 3A, 3B and 3C are graphs of four week in vivo data, glycosaminoglycan content, total GAG/dry weight (micrograms/mg) (FIG. 3A); hydroxyproline content, total hydroxyproline content/dry weight (micrograms/mg) (FIG. 3B); and ash weights, ash weight/dry weight (mg/mg) (FIG. 3C) for control and experimental.
FIG. 4 is a graph of radiodensitometry at twelve weeks post-operative implantation, radiodensity/opacity for control and experimental.
The disadvantages of autogenous bone grafts, including limited amount of bone and donor site morbidity, have prompted a search for a dependable bone graft substitute. Periosteum has been demonstrated to have cell populations that can be isolated in tissue culture and can form both cartilage and bone. Periosteal cells isolated under chondrogenic conditions and seeded in the polymer scaffolds have been used to repair articular cartilage defects. In the following example, periosteal cells cultured under osteogenic conditions were seeded into resorbable polymer scaffolds and used to repair critical size cranial bone defects.
Periosteum is a dense, white, fibrous covering around the surface of the bone not covered by articular cartilage. The periosteum consists of two layers. The outer fibrous layer is composed of connective tissue containing blood vessels, lymphatic vessels, and nerves that pass into the bone. The inner osteogenic layer contains elastic fibers, blood vessels, and osteoprogenitor cells and osteoblasts. The periosteum is essential for bone growth, repair and nutrition. It also serves as a point of attachment for ligaments and tendons.
Periosteum is obtained by surgically removing the periosteum from bone.
Periosteum consists of multipotent mesodermal cells. Periosteum has the capacity to form all varieties of connective tissue. The proliferative and bone-forming capacity of the osteogenic cells of the periosteal cambium layer has been confirmed in numerous studies. The periosteum culture conditions must be controlled to yield bone. Previous studies have established that transplanted periosteum differentiates based on the type of tissue or bone substrate onto which it is implanted; long bone appears to be better for inducing growth of bone as compared to calvarial bone. It is therefore critical for use of the periosteal cells to form bone to culture the cells under conditions leading to differentiation into bone, rather than cartilage, bone, and other tissues.
Periosteum is obtained by surgical removal of the tissue from the bone. The advantage of the procedure is that it does not require removal of bone per se. The periodsteum is dissected carefully using sharp periosteal elevators. Care is taken to ensure the cambium layer is removed completely. Confirmation of harvest can be accomplished by frozen or conventional histology.
The periosteum is digested using collagenase and trypsin as described by Cohn and Wong, In Skeletal Research ed. Simmons and Kunin, p. 3 (Academic Press, NY 1979). Alternatively, cell lines can be established by explant culture. Cells are cultured in Ham's F-12 medium (Gibco) at pH 7.6 supplemented with penicillin (100 U/cc), streptomycin sulfate (100 μg/cc), magnesium sulfate (200 μg/cc), glutamine (58.5 μg/cc) and fetal bovine serum at a final concentration of 12%. Culture medium is replaced three times per week. Cell passage is carried out by incubating monolayers for 5-10 minutes in calcium and magnesium-free Tyrode's solution containing 0.25% trypsin and replating the cells in fresh medium at one-third their confluent density.
Several techniques and materials, alone or in combination, can be employed to promote the osteoblastic phenotype of the periosteal cells, including high oxygen tension, addition of specific compounds or peptides, or specific matrices. In addition to specific matrix interactions, factors determining in vitro differentiation of the cells into bone include high oxygen tension, presence of steroid compounds, especially glucocorticoids such as dexamethasone and other steroidal antiinflammatories and cortisol, proteins and peptides such as calciferol and other enzymes enhancing calcification, beta-glycerolphosphate and other enzymes enhancing phosphorus deposition, prostaglandins such as prostaglandin E2, vitamins C and D and oxygen content. The preferred conditions are described in the following examples. Effective concentrations of the foregoing depend on whether the compounds are used alone or in combination, as well as the relative amounts. Represeitative concentrations include 0.1 μM dexamethasone, 40 ng calciferol/ml culture media, 0.1 μM cortisol, and 0.1 μM prostaglandin E2. Agents which enhance differentiation into cartilage, and which are therefore not advantageous for formation of bone, are known, for example, transforming growth factor beta 1 at concentrations up to 100 ng/ml. In vivo, even if initially induced in vitro to differentiate into bone, the substrate or site of implantation is the primary factor determining differentiation of the cells into bone.
Cells are examined and characterized by both phase contrast microscopy and light reflectance microscopy. Determination of osteoblast-like character is performed through a variety of methods. Osteocalcin production is measured using the method of Gundberg, Lian, and Gallop, Clin. Chem. Acta 128, 1 (1983). Monolayer cultures are washed and incubated in serum-free medium containing 10 mg/cc bovine serum albumin in the absence or presence of 10 nM 1,25(dihydroxy)vitaminD3 for 48 hours. The medium is analyzed by radioimmunoassay for osteocalcin at the end of the incubation period.
A number of assays can be used to evaluate the cells. For example, cAMP response to parathyroid hormone (PTH) is measured by incubated cultures near confluency with 1 mM isobutylmethylxanthine alone or in combination with 0.2 to 200 ng/cc of PTH with 1-10 ng/cc calcitonin for 10 minutes at 37° C. Incubations are halted by quickly transferring the cultures to ice, washing rapidly with ice-cold Tyrode's solution, and then adding ice-cold ethanol containing 2 mM HCL. After homogenizing the cells in ethanol for 5 seconds, the samples are dried at 100° C. and cAMP concentrations are measured by radioimmunoassay.
Alkaline phosphatase activity of the cell lysates is determined in a similar manner using the method of Luben, Wong and Cohn, Endocrinology 35, 778 (1983) with n-nitro-phenylphosphate as substrate. Collagen synthesis is analyzed according to Schwartz. Cultures are incubated for 48 hours in Minimal Essential Medium (Gibco) containing fetal bovine serum (10%), sodium ascorbate (50 microgram/ml), B-aminopropionitrile and tritiated proline (50 UCi/cc). Collagen from the culture medium and cell layer is isolated, treated with pepsin, and after the addition of carrier type I collagen, analyzed by SDS-PAGE under reducing and non-reducing conditions, as described by Neville, J. Biol. Chem. 246, 6326 (1971). Banks of carrier protein are identified by staining with Coomassie blue. The gels are then sliced into 25 equal segments each of which is monitored for radioactive content. Collagen distribution is calculated according to the method of Goldberg, et al., Biochem. Pharmacol. 29, 869 (1980).
Matrices for Implantation
The cells are seeded onto and into a matrix for implantation to repair the defect. In the preferred embodiment, the matrix is formed of a synthetic, biocompatible polymer, most preferably a biodegradable polymer, more preferably degrading by hydrolysis rather than enzymolysis. In other embodiments, the matrix is formed of a material such as hydroxyapatite or mixtures of hydroxyapatite and polymer, or tricalcium phosphate. Matrix can also be sterilized bone or a porous metal alloy. The matrix can be in the form of a fibrous or sponge like structure, or a hydrogel. The advantage of the biodegradable material is that the only material ultimately remaining in the patient is the bone. The term "bioerodible", or "biodegradable", as used herein refers to materials which are enzymatically or chemically degraded in vivo into simpler chemical species.
Over the last decade there has been a tremendous increase in applications for polymeric materials. These materials are well suited to implantation as they can serve as a temporary scaffold to be replaced by host tissue, degrade by hydrolysis to non-toxic products, and be excreted, as described by Kulkarni, et al., J. Biomedical Materials Research, 5, 169-81 (1971); Hollinger, J. O. and G. C. Battistone, "Biodegradable Bone Repair Materials, " Clinical Orthopedics and Related Research, 207, 290-305 (1986).
Either natural or synthetic polymers can be used to form the matrix, although synthetic polymers are preferred for reproducibility and controlled release kinetics. Synthetic polymers that can be used include bioerodible polymers such as poly(lactide) (PLA), poly(glycolic acid) (PGA), poly(lactide-co-glycolide) (PLGA), and other poly(alpha-hydroxy acids), poly(caprolactone), polycarbonates, polyamides, polyanhydrides, polyamino acids, polyortho esters, polyacetals, polycyanoacrylates and degradable polyurethanes, and non-erodible polymers such as polyacrylates, ethylene-vinyl acetate polymers and other acyl substituted cellulose acetates and derivatives thereof, non-erodible polyurethanes, polystyrenes, polyvinyl chloride, polyvinyl fluoride, poly(vinyl imidazole), chlorosulphonated polyolifins, polyethylene oxide, polyvinyl alcohol, and nylon. Although non-degradable materials can be used to form the matrix or a portion of the matrix, they are not preferred. Examples of natural polymers include proteins such as albumin, collagen, synthetic polyamino acids, and prolamines, and polysaccharides such as alginate, heparin, and other naturally occurring biodegradable polymers of sugar units.
Four polymers widely used in medical applications are poly(paradioxanone) (PDS), poly(lactic acid) (PLA), poly(glycolic acid) (PGA), and PLAGA copolymers. Copolymerization enables modulation of the degradation time of the material. By changing the ratios of crystalline to amorphous polymers during polymerization, properties of the resulting material can be altered to suit the needs of the application. These polymers, including poly(lactide-co-glycolic) acid (PLGA), have been used as polymer composites for bone replacement as reported by H. M. Elgendy, et al. "Osteoblast-like cell (MC3T3-E1) proliferation on bioerodible polymers: An approach towards the development of a bone-bioerodible polymer composite material, " Biomaterials, 14, 263-269 (1993). Substituted polyphosphazenes have been shown to support osteogenic cell growth, as reported by C. T. Laurencin, et al. "Use of polyphosphazenes for skeletal tissue regeneration, " J. Biom. Mater. Res., 27 (1993). Poly(organophosphazenes) are high molecular weight polymers containing a backbone of alternating phosphorus and nitrogen atoms. There are a wide variety of polyphosphazenes, each derived from the same precursor polymer, poly(dichlorophosphazene). The chlorine-substituted species can be modified by replacement of the chlorine atoms by different organic nucleophiles such as o-methylphenoxide along with amino acids. The physical and chemical properties of the polymer can be altered by adding various ratios of hydrolytic sensitive side chains such as ethyl glycinate, as described by C. W. R. Wade, et al. "Biocompatibility of eight poly(organophosphazenes), " in Organomet. Polym., C. E. Carraher, J. E. Sheats and C. U. Pitman, Jr., Eds., Academic Press, New York, 1978, pp. 283-288; and H. R. Allcock and T. J. Fuller, "Synthesis and Hydrolysis of Hexakis(imidazolyl)cyclotriphosphazene, " J. Am. Chem. Soc., 103, 2250-2256 (1981). This will affect the degradation of the polymer as an implantable and biodegradable material as well as vary the support of osteogenic cells for bone and tissue implants, as shown by Laruencin, et al. (1993).
PLA, PGA and PLA/PGA copolymers are particularly useful for forming the biodegradable matrices. PLA polymers are usually prepared from the cyclic esters of lactic acids. Both L(+) and D(-) forms of lactic acid can be used to prepare the PLA polymers, as well as the optically inactive DL-lactic acid mixture of D(-) and L(+) lactic acids. Methods of preparing polylactides are well documented in the patent literature. The following U.S. Patents, the teachings of which are hereby incorporated by reference, describe in detail suitable polylactides, their properties and their preparation: U.S. Pat. Nos. 1,995,970 to Dorough; 2,703,316 to Schneider; 2,758,987 to Salzberg; 2,951,828 to Zeile; 2,676,945 to Higgins; and 2,683,136; 3,531,561 to Trehu. PGA is the homopolymer of glycolic acid (hydroxyacetic acid). In the conversion of glycolic acid to poly(glycolic acid), glycolic acid is initially reacted with itself to form the cyclic ester glycolide, which in the presence of heat and a catalyst is converted to a high molecular weight linear-chain polymer. PGA polymers and their properties are described in more detail in Cyanamid Research Develops World's First Synthetic Absorbable Suture", Chemistry and Industry, 905 (1970).
The erosion of the matrix is related to the molecular weights of PLA, PGA or PLA/PGA. The higher molecular weights, weight average molecular weights of 90,000 or higher, result in polymer matrices which retain their structural integrity for longer periods of time; while lower molecular weights, weight average molecular weights of 30,000 or less, result in both slower release and shorter matrix lives. Poly(lactide-co-glycolide) (50:50), degrades in about six weeks following implantation.
All polymers for use in the matrix must meet the mechanical and biochemical parameters necessary to provide adequate support for the cells with subsequent growth and proliferation. The polymers can be characterized with respect to mechanical properties such as tensile strength using an Instron tester, for polymer molecular weight by gel permeation chromatography (GPC), glass transition temperature by differential scanning calorimetry (DSC) and bond structure by infrared (IR) spectroscopy, with respect to toxicology by initial screening tests involving Ames assays and in vitro teratogenicity assays, and implantation studies in animals for immunogenicity, inflammation, release and degradation studies.
These polymers are particularly useful in forming fibrous or sponge type matrices for implantation. Polymers can also be used to form hydrogels in which the cells are suspended and then implanted.
Another class of materials for making the matrix is hydroxyapatite, or a similar ceramic formed of tricalcium phosphate (TCP) or calcium phosphate (CaPO4).
Calcium hydroxyapatites occur naturally as geological deposits and in normal biological tissues, principally bone, cartilage, enamel, dentin, and cementum of vertebrates and in many sites of pathological calcifications such as blood vessels and skin. Synthetic calcium hydroxyapatite is formed in the laboratory either as pure Ca10 (PO4)6 (OH)2 or hydroxyapatite that is impure, containing other ions such as carbonate, fluoride, chloride for example, or crystals deficient in calcium or crystals in which calcium is partly or completely replaced by other ions such as barium, strontium and lead. Essentially none of the geological and biological apatites are "pure" hydroxyapatite since they contain a variety of other ions and cations and may have different ratios of calcium to phosphorous than the pure synthetic apatites.
In general, the crystals of pure synthetic apatites, geological apatites and many impure synthetically produced apatites are larger and more crystalline than the biological crystals of bone, dentin, cementum and cartilage. The crystals of bone, dentin and cementum are very small, irregularly shaped, very thin plates whose rough average dimensions are approximately 10 to 50 angstroms in thickness, 30 to 150 angstroms in width, and 200 to 600 angstroms in length. The synthetic materials are highly diverse, as reported in the literature. For example, the characterization of four commercial apatites was reported by Pinholt, et al., J. Oral Maxillofac. Surg. 50(8), 859-867 (August 1992); J. Cariofac. Surg. 1(3), 154-160 (July 1990) reports on a protein, biodegradable material; Pinholt, et al., Scand. J. Dent. Res. 99(2), 154-161 (April 1991) reports on the use of a bovine bone material called Bio-Oss™; Friedman, et al., Arch. Otolaryngol. Head Neck Surg. 117(4), 386-389 (April 1991) and Costantino, et al., Arch. Otolaryngol. Head Neck Surg. 117(4), 379-384 (April 1991) report on a hydroxyapatite cement; Roesgen, Unfallchirurgle 16(5), 258-265 (October 1990), reports on the use of calcium phosphate ceramics in combination with autogenie bone; Ono, et al., Biomaterials 11(4), 265-271 (May 1990) reports on the use of apatite-wollastonite containing glass ceramic granules, hydroxyapatite granules, and alumina granules; Passuti, et al., Clin. Orthop. 248, 169-176 (Nov. 1989) reports on macroporous calcium phosphate ceramic performance; Harada, Shikwa-Gakuho 89(2), 263-297 (1989) reports on the use of a mixture of hydroxyapatite particles and tricalcium phosphate powder for bone implantation; Ohgushi, et al., Acta Orthop. Scand. 60(3), 334-339 (1989) reports on the use of porous calcium phosphate ceramics alone and in combination with bone marrow cells; Pochon, et al., Z-Kinderchir. 41(3), 171-173 (1986) reports on the use of beta-tricalcium phosphate for implantation; and Glowacki, et al., Clin. Plast. Surg. 12(2), 233-241 (1985), reports on the use of demineralized bone implants.
As used herein, all of these materials are generally referred to as "hydroxyapatite". In the preferred form, the hydroxyapatite is particles having a diameter between approximately ten and 100 microns in diameter, most preferably about 50 μ in diameter.
Calcium phosphate ceramics can be used as implants in the repair of bone defects because these materials are non-toxic, non-immunogenic, and are composed of calcium and phosphate ions, the main constituents of bone (Jarcho, 1981; Frame, J. W., "Hydroxyapatite as a biomaterial for alveolar ridge augmentation, " Int. J. Oral Maxillofacial Surgery, 16, 642-55 (1987); Parsons, et al. "Osteoconductive Composite Grouts for Orthopedic Use, " Annals N.Y. Academy of Sciences, 523, 190-207 (1988)). Both tricalcium phosphate (TCP) Ca3 (PO4)2 ! and hydroxyapatite (HA) Ca10 (PO4)6 (OH2 ! have been widely used. Calcium phosphate implants are osteoconductive, and have the apparent ability to become directly bonded to bone. As a result, a strong bone-implant interface is created.
Calcium phosphate ceramics have a degree of bioresorbability which is governed by their chemistry and material structure. High density HA and TCP implants exhibit little resorption, while porous ones are more easily broken down by dissolution in body fluids and resorbed by phagocytosis. However, TCP degrades more quickly than HA structures of the same porosity in vitro. HA is relatively insoluble in aqueous environments. However, the mechanical properties of calcium phosphate ceramics make them ill-suited to serve as a structural element under load bearing circumstances. Ceramics are not preferred since they are brittle and have low resistance to impact loading.
For an organ to be constructed, successfully implanted, and function, the matrices must have sufficient surface area and exposure to nutrients such that cellular growth and differentiation can occur prior to the ingrowth of blood vessels following implantation. The time required for successful implantation and growth of the cells within the matrix is greatly reduced if the area into which the matrix is implanted is prevascularized. After implantation, the configuration must allow for diffusion of nutrients and waste products and for continued blood vessel ingrowth as cell proliferation occurs.
The organization of the tissue may be regulated by the microstructure of the matrix. Specific pore sizes and structures may be utilized to control the pattern and extent of fibrovascular tissue ingrowth from the host, as well as the organization of the implanted cells. The surface geometry and chemistry of the matrix may be regulated to control the adhesion, organization, and function of implanted cells or host cells.
In the preferred embodiment, the matrix is formed of polymers having a fibrous structure which has sufficient interstitial spacing typically in the range of 100 to 300 microns. A microporous structure (pores 205 μm in diameter) has been shown to be too small to permit the ingrowth of cells, as reported by Friedlander, G. E. and V. M. Goldberg, Bone and Cartilage Allografis, Park Ridge: American Academy of Orthopedic Surgeons, 1991; Jarcho, M. "Calcium Phosphate Ceramics as Hard Tissue Prosthetics, " Clinical Orthopedics and Related Research, 157, 259-78 (1981). As used herein, "fibrous" includes one or more fibers that is entwined with itself, multiple fibers in a woven or non-woven mesh, and sponge like devices.
The overall, or external, matrix configuration is dependent on the tissue which is to reconstructed or augmented. These are readily determined by the surgeon based on the defect to be corrected.
Polymers that can form ionic hydrogels which are malleable can also be used to support the cells. Injecting a suspension of cells in a polymer solution may be performed to improve the reproducibility of cell seeding throughout a device, to protect the cells from shear forces or pressure induced necrosis, or to aid in defining the spatial location of cell delivery. The injectable polymer may also be utilized to deliver ells and promote the formation of new tissue without the use of any other matrix. In a preferred embodiment, the hydrogel is produced by cross-linking the ionic salt of a polymer with ions, whose strength increases with either increasing concentrations of ions or polymer. The polymer solution is mixed with the cells to be implanted to form a suspension, which is then injected directly into a patient prior to hardening of the suspension. The suspension subsequently hardens over a short period of time due to the presence in vivo of physiological concentrations of ions such as calcium in the case where the polymer is a polysaccharide such as alginate.
A hydrogel is defined as a substance formed when an organic polymer (natural or synthetic) is cross-linked via covalent, ionic, or hydrogen bonds to create a three-dimensional open-lattice structure which entraps water molecules to form a gel. Examples of materials which can be used to form a hydrogel include polysaccharides such as alginate, polyphosphazenes, and polyacrylates such as hydroxyethyl methacrylate (HEMA), which are crosslinked ionically, or block copolymers such as Pluronics™ or Tetronics™, polyethylene oxide-polypropylene glycol block copolymers which are crosslinked by temperature or pH, respectively. Other materials include proteins such as fibrin, polymers such as polyvinylpyrrolidone, hyaluronic acid and collagen.
In general, these polymers are at least partially soluble in aqueous solutions, such as water, buffered salt solutions, or aqueous alcohol solutions, that have charged side groups, or a monovalent ionic salt thereof. Examples of polymers with acidic side groups that can be reacted with cations are poly(phosphazenes), poly(acrylic acids), poly(methacrylic acids), copolymers of acrylic acid and methacrylic acid, poly(vinyl acetate), and sulfonated polymers, such as sulfonated polystyrene. Copolymers having acidic side groups formed by reaction of acrylic or methacrylic acid and vinyl ether monomers or polymers can also be used. Examples of acidic groups are carboxylic acid groups, sulfonic acid groups, halogenated (preferably fluorinated) alcohol groups, phenolic OH groups, and acidic OH groups. Examples of polymers with basic side groups that can be reacted with anions are poly(vinyl amines), poly(vinyl pyridine), poly(vinyl imidazole), and some imino substituted polyphosphazenes. The ammonium or quaternary salt of the polymers can also be formed from the backbone nitrogens or pendant imino groups. Examples of basic side groups are amino and imino groups.
Alginate can be ionically cross-linked with divalent cations, in water, at room temperature, to form a hydrogel matrix. Due to these mild conditions, alginate has been the most commonly used polymer for hybridoma cell encapsulation, as described, for example, in U.S. Pat. No. 4,352,883 to Lim. In the Lim process, an aqueous solution containing the biological materials to be encapsulated is suspended in a solution of a water soluble polymer, the suspension is formed into droplets which are configured into discrete microcapsules by contact with multivalent cations, then the surface of the microcapsules is crosslinked with polyamino acids to form a semipermeable membrane around the encapsulated materials.
The polyphosphazenes suitable for cross-linking have a majority of side chain groups which are acidic and capable of forming salt bridges with di- or trivalent cations. Examples of preferred acidic side groups are carboxylic acid groups and sulfonic acid groups. Hydrolyrically stable polyphosphazenes are formed of monomers having carboxylic acid side groups that are crosslinked by divalent or trivalent cations such as Ca2+ or Al3+. Polymers can be synthesized that degrade by hydrolysis by incorporating monomers having imidazole, amino acid ester, or glycerol side groups. Bioerodible polyphosphazenes have at least two differing types of side chains, acidic side groups capable of forming salt bridges with multivalent cations, and side groups that hydrolyze under in vivo conditions, e. g., imidazole groups, amino acid esters, glycerol and glucosyl.
The water soluble polymer with charged side groups is crosslinked by reacting the polymer with an aqueous solution containing multivalent ions of the opposite charge, either multivalent cations if the polymer has acidic side groups or multivalent anions if the polymer has basic side groups. The preferred cations for cross-linking of the polymers with acidic side groups to form a hydrogel are divalent and trivalent cations such as copper, calcium, aluminum, magnesium, strontium, barium, and tin, although di-, tri- or tetra-functional organic cations such as alkylammonium salts, e. g., R3 N+ --+NR3 can also be used. Aqueous solutions of the salts of these cations are added to the polymers to form soft, highly swollen hydrogels and membranes. The higher the concentration of cation, or the higher the valence, the greater the degree of cross-linking of the polymer. Concentrations from as low as 0.005M have been demonstrated to cross-link the polymer. Higher concentrations are limited by the solubility of the salt. The preferred anions for cross-linking of the polymers to form a hydrogel are divalent and trivalent anions such as low molecular weight dicarboxylic acids, for example, terepthalic acid, sulfate ions and carbonate ions. Aqueous solutions of the salts of these anions are added to the polymers to form soft, highly swollen hydrogels and membranes, as described with respect to cations.
A variety of polycations can be used to complex and thereby stabilize the polymer hydrogel into a semi-permeable surface membrane. Examples of materials that can be used include polymers having basic reactive groups such as amine or imine groups, having a preferred molecular weight between 3,000 and 100,000, such as polyethylenimine and polylysine. These are commercially available. One polycation is poly(L-lysine), examples of synthetic polyamines are: polyethyleneimine, poly(vinylamine), and poly(allyl amine). There are also natural polycations such as the polysaccharide, chitosan. Polyanions that can be used to form a semi-permeable membrane by reaction with basic surface groups on the polymer hydrogel include polymers and copolymers of acrylic acid, methacrylic acid, and other derivatives of acrylic acid, polymers with pendant SO3 H groups such as sulfonated polystyrene, and polystyrene with carboxylic acid groups.
Seeding and Implantation of the Matrix
In the preferred embodiment, the cells are seeded onto and into the matrix, then cultured for a time dependent on the matrix material which is employed. For example, when biodegradable polymeric matrices are used, cells will typically be cultured for between one and five weeks. Cells cultured on matrices formed of a material such as hydroxyapatite can be cultured for between one and ten weeks. The matrix is then surgically implanted at the site of the defect to be corrected, using standard surgical techniques.
In the case of a hydrogel, the polymer is dissolved in an aqueous solution, preferably a 0.1M potassium phosphate solution, at physiological pH, to a concentration forming a polymeric hydrogel, for example, for alginate, of between 0.5 to 2% by weight, preferably 1%, alginate. The isolated cells are suspended in the polymer solution to a concentration of between 1 and 50 million cells/ml, most preferably between 10 and 20 million cells/ml. This is then injected into the site where the repair is needed.
The present invention will be further understood by reference to the following non-limiting examples.
Materials and Methods:
Isolation and Culture of Periosteal Derived mesenchymal Stem Cells:
Periosteal tissue was isolated from the proximal tibiae of adult New Zealand White (NZW) rabbits. A 5×10 mm rectangular incision was made to expose the bone, and the periostea was then separated from the underlying bone using sharp periosteal elevators. Care was taken to insure the harvesting of the cambium layer. The tissue was immediately placed in Tyrodes buffer and under sterile conditions diced into small (1 mm) pieces. Explants were placed in Hams F-12 complete tissue culture medium. A second set of explants were cultured in medium supplemented with 0.1 μM dexamethasone. Explants were checked for outgrowth daily and fed fresh medium every other day. Cell lines were serial passaged no more than twice. After cell number amplification was completed, they were trypsinized, washed, and counted. The cells were labeled in log phase of growth in vitro with the nucleic acid analog bromodeoxyuridine (BrDU).
Assembly of Cell Polymer/Tissue Construct Grafts:
The cell pellet was resuspended in 200 μl of medium and then seeded into polyglycolic acid (PGA) non-woven fiber scaffolds (15 mm diam.×2 mm thick, pore size 200 μM) at a density of 4×106 cells/graft. Tissue constructs were allowed to settle for 9 hours in a humidified incubator at 37° C. to allow cells to attach to the scaffold. Assembled tissue constructs were then further cultured for a period of two weeks prior to implantation. In vitro assays of osteoblastic phenotype, including vitamin D stimulation of osteocalcin, and glycosaminoglycan content were performed.
Surgical Procedure:
A total of 24 adult male NZW rabbits were used in this study and divided into control and experimental groups. Rabbits were anesthetized with ketamine/xylazine. Under sterile conditions, a mid-sagittal incision was made and flaps were retracted in a subperiosteal plane, exposing the parietal bone. A 15 mm diameter calvarial defect (critical size defect in the rabbit) was made unilaterally in the parietal bone using a saline cooled trephination drill. In the experimental animals, defects were repaired using a PGA implant seeded with periosteal cells. In the control animals, an untreated PGA implant was used to repair the defects.
Rabbits were divided into groups and euthanized at 4 and 12 weeks after surgery. After sacrifice, the defect site was radiographed and then cut into sagittal halves. One half was fixed in formalin and processed for hematoxylin and eosin (H & E) histology and immunofluorescence studies. The other half was assayed for glycosaminoglycan content, collagen content, and ash weight analysis.
Osteocalcin Assay:
In vitro constructs were incubated with vitamin D (10-2 M) for 72 hours. The supernatant was collected, frozen, and the quantity of osteocalcin induced by exposure to vitamin D was determined using a radioimmunoassay.
Total Glycosaminolglycan (GAG) Assay:
The presence of GAG in the repair tissue was determined by the improved DMB method of Farndale. Tissue samples were harvested, dried, weighed, and then digested in a papain-versene solution overnight at 65° C. Aliquots of the tissue digest were then mixed with the dye dimethylene blue (DMB) and read in a spectrophotometer at 535 Å. Chondroitin sulfate was used as a standard for instrument calibration.
Total Collagen Content Assay:
Collagen content in repair sites was assayed using the method of Woessner. Briefly, samples were dried, weighed, and hydrolyzed in 12N HCl at 105° C. for 16 to 24 hours. Samples were mixed with a solution containing Chloramine T heated at 60° C. for 30 minutes and read in a spectrophotometer at 550 nm. Appropriate standards were used for quantitative determination.
Ash Weight Determination:
The amount of bone mineral present at the repair site was determined using a standard ashing method. Replicates of repair tissue from the defect site were harvested, dried at 100° C. for two days, and then weighed. The samples were then placed in an ashing furnace overnight and the sample was then reweighed. Percent bone mineral was then calculated using total dry weight and ash weight.
Specimens were fixed in buffered formalin and then decalcified in EDTA, embedded in paraffin, and section were cut at 5 μm in thickness. Slides were stained with H&E and Mallory trichrome.
Cell Labeling and Localization:
Cultured periosteal cells were labeled in log phase of growth in vitro with the nucleic acid analog bromodeoxyuridine (BrDU). Localization was performed using a fluorescein tagged monoclonal antibody and observed under fluorescent microscopy.
Quantitative Radiodensitometry:
Radiographs were taken of the defect site using Kodak x-omat TL film at 50 kV/200 mAs. The area of the defect site on the film was scanned on a Biorad densitometer. Intensity levels were set using normal calvarial bone and blank film.
In Vitro:
Periosteal cells grew form the explanted tissue within four days after harvest and continued to divide with or without the presence of dexamethasone. However, GAG content was significantly enhanced in constructs cultured with dexamethasone at all time points studied, as shown by FIG. 1. The GAG content did fluctuate with time and did not display any trends. Osteocalcin was produced by the periosteal cells regardless of the presence of dexamethasone. Osteocalcin content increased as a function of time in culture, as shown by FIG. 2. Histological appearance of these calls was fibroblastoid with a spindle morphology in monolayer culture. The morphology was altered when placed into the three dimensional scaffold to a more stellate shape usually with several processes.
In Vivo:
At four weeks, upon gross examination, the experimental defect sites were filled with a homogenous stiff membrane, whereas the control defect sites were occupied by soft fibrous tissue. H & E histology showed scattered islands of bone in the experimental defects and fibrous tissue in the control defects. Glycosaminoglycan content, collagen content, and ash weight were significantly increased in the experimental defect sites compared with the control defect sites, as shown by FIGS. 3A, 3B and 3C.
At 8 weeks, although gross and histological examination revealed areas of bone in both the experimental and control defects, there was more bone in the experimental than control defects, there was more bone in the experimental defects. Quantitative analysis of the radiographs (radiodensitometry) showed significantly increased radiopacity in the experimental defects compared with the control defects, as shown by FIG. 4. BrDU fluorescent label was detected in the newly formed bone in the experimental defects confirming the contribution of the cultured periosteal cells to this bone formation.
Rabbits were divided into groups and sacrificed at 4 and 12 weeks postoperatively. Repaired defect sites were then studied histologically, biochemically, and radiographically. In vitro analysis of the cultured periosteal cells indicated an osteoblastic phenotype with production of osteocalcin upon stimulation with Vitamin D, as well as increased amounts of glycosaminoglycan. In vivo results at 4 weeks showed islands of bone in the experimental defects, whereas the control defects were filled with fibrous tissue. Glycosaminoglycan content, collagen content, and ash weight were significantly increased in the experimental defects compared with the control defects. At 8 weeks, radiodensitometry demonstrated significantly increased bone formation in the experimental defects compared with the control defects. BrDU fluorescent label was detected in the newly formed bone in the experimental defects, confirming the contribution of the cultured periosteal cells to this bone formation.
Modifications and variations of the present invention will be obvious to those skilled in the art from the foregoing detailed description. Such modifications as to methods for obtaining periosteal cells, culturing the cells, seeding and implanting of a matrix to form new bone or repair bone defects are intended to come within the scope of the appended claims.
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|4 Mar 2009
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|Cohesive and compression resistant demineralized bone carrier matrix
|4 Jun 2009
|29 Oct 2009
|Musculoskeletal Transplant Foundation
|Allograft bone composition having a gelatin binder
|31 Jul 2007
|18 Feb 2010
|Bioform Medical, Inc.
|Implantation Compositions for Use in Tissue Augmentation
|14 Ago 2009
|4 Mar 2010
|The Government of the U.S.A, as represented by the Department of Veterans Affairs
|Methods of making a transplantable bone repair system using multipotent stem cells
|3 Sep 2009
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|Beijing Allgens Medical Science & Technology Co., Ltd.
|Hydroxyapatite ceramic for spinal fusion device
|29 Dic 2009
|22 Abr 2010
|Bioform Medical, Inc.
|Tissue Augmentation Material and Method
|3 Abr 2008
|1 Jul 2010
|The Cleveland Clinic Foundation
|Enrichment of Tissue-Derived Adult Stem Cells Based on Retained Extracellular Matrix Material
|28 May 2010
|23 Sep 2010
|Bioform Medical, Inc.
|Tissue Augmentation Material And Method
|17 Dic 2008
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|Methods of preparing a virtual dentition model and fabricating a dental retainer therefrom
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|Merz Aesthetics, Inc.
|Tissue Augmentation Material And Method
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|Treatment of bony defects with osteoblast precursor cells
|21 May 1999
|25 Nov 1999
|The Cleveland Clinic Foundation
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|17 Feb 2000
|Cleveland Clinic Foundation
|Apparatus and methods for preparing an implantable graft
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|24 Feb 2000
|Cambridge Scientific, Inc.
|Osteoinduction of cortical bone allografts by coating with biopolymers seeded with recipient periosteal bone cells
|6 Nov 2000
|18 Abr 2002
|Massachusetts Institute Of Technology
|Cell delivery using controllably degradable mesh-gel constructs
|4 Feb 2004
|24 Feb 2005
|Cambridge Polymer Group
|Systems and methods for controlling and forming polymer gels
|20 Mar 2012
|27 Sep 2012
|Endo Pharmaceuticals Inc.
|Urethral anastomosis device and method
|17 Sep 2013
|27 Mar 2014
|Endo Pharmaceuticals Inc.
|Urethral anastomosis device
|10 Mar 2014
|2 Oct 2014
|Endo Pharmaceuticals Inc.
|Urethral anastomosis device
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Sports Medicine
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Recent findings published in the American Journal of Public Health showed that students experienced greater academic dysfunction 1 week after concussion compared with students who experienced extremity injuries. However, 1 month after injury, there was no difference between the groups.
"Most students who play sports are not going to become professional athletes," Erin B. Wasserman, PhD, at the Matthew Gfeller Sport-Related Traumatic Brain Injury Research Center, University of North Carolina at Chapel Hill, said in a press release. "But, they will need to continue with school and prepare for a career."
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If you've been on a Birthright Israel trip, you can do something unforgettable this Passover: Host your friends for your very own Passover Seder. Share your favorite traditions, and create new ones. Invite your Birthright Israel friends, or friends who have never experienced a Seder before. Cook your grandma's brisket, or invent your own menu. It's Passover, your way. We'll make it easy by giving you all the resources you need!
The Birthright Israel Foundation is committed to protecting the privacy of our donors
Birthright Israel Foundation is listed by the IRS as a 501 (c)(3) non-profit. All
donations are tax deductible to the fullest extent of the law.


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My apologies if this has been posted before but I think you'll all agree this is important for cycling... not just in Britain. I've repeated this post in "Road". Please bump this to keep it up the top...
Mods: Are you able to "stickie" this on all forums?
This from Carlton Rieds site QuickRelease.tv - hope you don't mind me coping your txt Carlton
You may not live in Britain, but you could help us win $100m to transform 79 communities across the UK. The BIG Lottery Fund has unveiled the final shortlist for the Peoples £50 Million Lottery Giveaway. The contest will be a public vote which will see one project of national importance win a grant of £50m. The Sustrans Connect2 project is the only national project out of four finalists.
Sustrans stands a stunningly good chance against what are worthy but highly localised projects. Sign up on the Connect2 website to be reminded to vote online from November 26th onwards.
Click here http://www.sustransconnect2.org.uk/ to sign up for an email reminder to vote


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Senigallia Cemetery. The Old Part.
Mauro Taraborelli
Senigallia cemetery is a monumental cemetery that dates back to the second half of the 19th century. It covers the slopes of one of the hills that surround the town and walking trough its rows is of visual and historic interest.
This book is a photographic journey in the old part of the Senigallia cemetery, presented through four visual themes: colombarium, gates, flowers and plants, decorations.
Parallel texts: English and Italian.
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Senigallia Mare Collina
Published May 27, 2013
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It's been a while. Like, a long while.
Recently I've been going through my inbox and clearing everything out, although I think I've gone through enough to be able to just say a quick "hey, I'm alive." I only have 320 notifications to look through (but 200 of them are from one person, so)
I make no promises that I'll be active because I broke all the ones from before, but I've actually logged on multiple times these past few weeks, which is an accomplishment. Of course, when summer classes start, that'll probably end...
But this journal's here as an update or to catch up -- SO if anyone wants to talk, be my guest in the comments. I'll probably reply somewhere from 2 days to 2 years from now - you never know.
Also, I kind of want a different name, but I don't have the points and I don't have a name in mind. *shrug* we'll see where that goes.
(This journal had no point, did it?)


text:
Day #1 - Recap - Nikon V2
Started Oct 24, 2012 | Discussions thread
Shop cameras & lenses ▾
Daniel Oh • Regular Member • Posts: 468
Re: Day #1 - Recap - Nikon V2
I hope Nikon will listen to you.
Personally I would prefer to have articulated LCD also but I am sure there are a bunch of people who don't want one.
The price does not bother me. I know how to wait and most mirrorless cameras prices have been dropping up to 50% within 1-2 years (Nikon J1, V1, Olympus EPL2, EP2, EPM1, Sony 5N, Panasonic G2F etc). I may get a V2 body for christmas in 2013 for under $500 ...
-- hide signature --
Daniel Oh
ps. I truly believe photography as a hobby is defined by the pleasure you get from the activity. It does not matter what equipment or results. I am a hobbyist and a very awful photographer (snapshooter is a better name) but a very happy one.
Daniel Oh's gear list:Daniel Oh's gear list
Pentax Optio W60 Samsung EX2F Nikon D700 Nikon 1 J1 Nikon 1 V2 +13 more
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|Savard: I want to finish my career in Boston
|09.08.09 at 2:01 pm ET
Marc Savard and Zdeno Chara were the two building blocks for Bruins GM Peter Chiarelli when the hockey executive first came onto the scene in Boston, and the 32-year-old center — entering the final year of a four-year deal he signed on July 1, 2006 — said that he's hoping to sign another deal allowing him to finish his hockey career in Boston.
Savard was the B's leading scorer with 25 goals and 63 assists last season, and has blossomed into an All-Star player under coach Claude Julien. Entering the final year of his $5 million per year deal with a great deal of financial uncertainty in the NHL's future, Savard isn't bothered by the unknown and is simply focused on driving the B's toward a Stanley Cup.
Savard said he's fully recovered from the left knee injury suffered in the playoffs against the Carolina Hurricanes, and he skated "10 or 15 times" before hitting the practice ice with his teammates at Ristuccia Arena for the first time on Tuesday morning.
"It's a great city and I've enjoyed my time here so far," said Savard, from the Bruins Foundation's annual golf tournament at The International in Bolton, MA. "It's a place that I'd like to finish if the chance comes and I'm excited to get the season going. Things keep getting better and our team keeps getting better, so that only helps everybody when that happens. I want to stay here. This is a place I love. I love the people. I love the fans. This is where I want to be.
"I'm not worried about the contract at all. (Peter and I) have a good relationship. Ever since I came to Boston I've given everything I had and if things work out well — and I think they will — then I'm going to be here for a long, long time."
Savard was also visibly peeved when asked about getting snubbed by Hockey Canada when team officials announced the lineup for their Olympic Team Orientation Camp this summer. The crafty centerman was conspicuously absent from an admittedly talented roster of players, and that's not sitting well with the two-time All-Star. Savard said that he shared an agent, Larry Kelly, with Steve Yzerman, one of the hockey minds charged with constructing the Canadian National Team, and that private campaigning with the Team Canada Executive Director for an invitation didn't quite work for the skilled B's pivot.
Savard has instead decided to turn the first half of the 2009-10 hockey season into a resume tape for Team Canada to watch what they might be missing out in Vancover come February. Though he was on the driving range at the International and excited for a day of golf with his teammates, Savard couldn't hide the sting of disappointment at being left off the squad.
"I was pretty upset about it," said Savard, who then cast his head down toward the ground as he talked about it. "I feel that I had a chance to at least go to the camp. I didn't really come out and say anything. I had a lot of calls for a couple weeks after that. It's something I didn't want to talk about. I was pretty mad about it. I've had to prove myself over and over again. I'm hoping to get off to a good start. I still haven't counted myself out, so I guess that's all that matters.
"I'm going to go out and do what I do…try to prove somebody else wrong. I'm worried about the Bruins and winning games. I didn't really talk to anybody (behind the scenes) but my agent had Yzerman as a player and he did most of the talking. I don't know what happened, but I have to just keep trying to prove them wrong and have a good start to the season."
|Lucic invited, Savard snubbed for Team Canada camp
|07.02.09 at 12:44 pm ET
It was a dream come true for Milan Lucic when the 20-year-old hulking winger found out he was among the 46 names invited to the Team Canada Olympic orientation/tryout camp this summer. The Vancouver, B.C. native had a career-best 42 points in 72 games for the Bruins during his second season in the NHL during 2008-09, and made a name around the league with his punishing body checks and physical presence at such a tender young age.
Marc Savard, on the other hand, was a notable name omitted from the preliminary Olympic list after getting plenty of support for the team during the NHL season after continuing to develop his two-way game under B's head coach Claude Julien. Team Canada is loaded with talented centers among the 46 invitees, but most hockey observers would be hard-pressed to explain how St. Louis Blues center Andy McDonald is more Olympics-worthy than a two-time All-Star in Savard. Savard has averaged 89 points a season over the last four years and was a career-best +25 with the Bruins during a breakout year for the team.
Team Canada's camp is scheduled from August 24-27 at the Pengrowth Saddledome in Calgary, Alberta, and 46 players were identified Thursday morning as candidates for upcoming international events in the 2009-10 season: the 2010 Olympic Winter Games and 2010 IIHF World Championship.
The 46 invitees include: CANADA'S MEN'S NATIONAL TEAM ORIENTATION CAMP ROSTER
Goaltenders (5):
Martin Brodeur (Montreal, Que./New Jersey, NHL), Marc-André Fleury
(Sorel, Que./Pittsburgh, NHL), Roberto Luongo (Montreal, Que./Vancouver,
NHL), Steve Mason (Oakville, Ont./Columbus, NHL), Cam Ward (Sherwood
Park, Alta./Carolina, NHL)
Defensemen (16)
François Beauchemin (Sorel, Que./Anaheim, NHL), Jay Bouwmeester
(Edmonton, Alta./Calgary, NHL), Dan Boyle (Ottawa, Ont./San Jose, NHL),
Brent Burns (Ajax, Ont./Minnesota, NHL), Drew Doughty (London, Ont./Los
Angeles, NHL), Mike Green (Calgary, Alta./Washington, NHL), Dan Hamhuis
(Smithers, B.C./Nashville, NHL), Duncan Keith (Penticton, B.C./Chicago,
NHL), Scott Niedermayer (Cranbrook, B.C./Anaheim, NHL), Dion Phaneuf
(Edmonton, Alta./Calgary, NHL), Chris Pronger (Dryden,
Ont./Philadelphia, NHL), Robyn Regehr (Rosthern, Sask./Calgary, NHL),
Stéphane Robidas (Sherbrooke, Que./Dallas, NHL), Brent Seabrook
(Tsawwassen, B.C./Chicago, NHL), Marc Staal (Thunder Bay, Ont./N.Y.
Rangers, NHL), Shea Weber (Sicamous, B.C./Nashville, NHL)
Forwards (25)
Jeff Carter (London, Ont./Philadelphia, NHL), Dan Cleary (Carboneau,
N.L./Detroit, NHL), Sidney Crosby (Cole Harbour, N.S./Pittsburgh, NHL),
Shane Doan (Halkirk, Alta./Phoenix, NHL), Simon Gagné (Ste-Foy,
Que./Philadelphia, NHL) , Ryan Getzlaf (Regina, Sask./Anaheim, NHL), Dany
Heatley (Calgary, Alta./Ottawa, NHL), Jarome Iginla (St. Albert,
Alta./Calgary, NHL), Vincent Lecavalier (Ãle-Bizard, Que./Tampa Bay,
NHL), Milan Lucic (Vancouver, B.C./Boston, NHL), Patrick Marleau
(Aneroid, Sask./San Jose, NHL), Andy McDonald (Strathroy, Ont./St.
Louis, NHL), Brenden Morrow (Carlyle, Sask./Dallas, NHL), Rick Nash
(Brampton, Ont./Columbus, NHL), Corey Perry (Peterborough, Ont./Anaheim,
NHL), Michael Richards (Kenora, Ont./Philadelphia, NHL), Derek Roy
(Rockland, Ont./Buffalo, NHL), Joe Sakic (Burnaby, B.C./Colorado, NHL),
Patrick Sharp (Thunder Bay, Ont./Chicago, NHL), Ryan Smyth (Banff,
Alta./Colorado, NHL), Martin St-Louis (Laval, Que./Tampa Bay, NHL), Eric
Staal (Thunder Bay, Ont./Carolina, NHL), Jordan Staal (Thunder Bay,
Ont./Pittsburgh, NHL), Joe Thornton (St. Thomas, Ont./San Jose, NHL),
Jonathan Toews (Winnipeg, Man./Chicago, NHL)
|Bruins sign Steve Begin to a one-year deal worth $850K
|07.01.09 at 6:48 pm ET
The Bruins continued their flurry of July 1 activity by signing former Montreal Canadiens agitator Steve Begin to a one-year deal worth $850,000 on late Wednesday afternoon. The 31-year-old Begin registered 12 points (7 goals, 5 assists) and 42 penalty minutes in 63 games combined for the Canadiens and Dallas Stars last season, and is being brought into the B's fold to provide a little extra grit and a lot of extra nasty into the bottom two lines next season.
For his part, Begin said that his experience playing for Julien with the Habs — where he enjoyed his best season under Julien with the Habs while scoring 11 goals and 12 assists along with 113 PIMs in 76 games — was one of the big things that attracted him to Boston.
"I know Claude Julien. He coached me in Montreal for two years and I know Claude, he's a great coach. I know that Boston has some great players, so this was really an easy choice for me," said Begin. "It's going to be fun to have those guys on my side now. With Montreal those were games were always big (against the Bruins) so it's going to be fun to come to the other side now.
"One day you're on the one team and then another day you're on the other team. Now I'm going to be on the right side (of the rivalry) now. (With Claude) he listens to the players and he knows how to play you and how to use you. I think if you look at (Michael) Ryder, a year ago in Montreal people thought he was done and now he's playing for Claude and he had his best season last year. He gave me a lot of ice time and played me a lot."
Being cast off by the Canadiens during the rough patch last might have also played into the decision to don the Spoked B as well, but Begin wasn't biting on that one.
Bruins fans will remember Begin as one of Montreal's hatchet men during the 2007-08 season, and the Quebec native memorably cross-checked Marc Savard from behind and broke a bone in the center's back just prior to the playoffs — a questionable move that opened the door for David Krejci to finally establish himself as an NHL player. The B's didn't really have an "agitator" last season that provoked and got under the skin of the opposition, and that's a role that the 6-foot, 193-pound Begin can play with aplomb.
"He's a real physical guy. He's not huge, but he's big enough and he makes up for it in the way that he plays," said Bruins GM Peter Chiarelli. "He's got history with Claude and he's just a tremendous, tremendous competitive player and person. He kills penalties, will fill a role and had some energy so I'm excited to get him."
Bruins GM Peter Chiarelli confirmed the signing during a Wednesday night conference call and said that Begin effectively takes the roster spot formerly occupied by veteran center Stephane Yelle, who did a solid job with faceoffs and killing penalties on the fourth line in Boston last season.
With Steve Montador's signing in Buffalo and the admission that Yelle's term with the Bruins is over — along with Wednesday night's announcement that defenseman Johnny Boychuk was signed to a one-way deal with the Bruins — that means at least two new faces will be into the Black and Gold mix this season.
"I guess if you look at it, Steve effectively replaces Stephane (Yelle) if you want to get to the nitty gritty. If you want to look at him and how he plays, he's a versatile player, he's a useful player and he's a gritty player," said Chiarelli. "So he's a guy you can slide a little bit up the lineup, he's a guy that can kill penalties and he's a guy that will wear the emblem on his sleeve. There's a lot of good things about him. Over the few years I've been here we've back-filled with these types of players, and we expect Steve to be one of those."
Begin will join Byron Bitz and Shawn Thornton on a potential fourth line grouping that could become a thoroughly enjoyable trio of physical, gritty forwards capable of punishing and intimidating opposing lines in an Eastern Conference that seems to be getting bigger and nastier with each passing day.
"I like to finish my checks and I like to chip in once in a while, but it's a bonus when I do. Mostly I bring a lot of energy and I'm a team guy. I'm working hard, skating hard and finishing my checks, and it's all of the things that you saw Boston do with guys like (Shawn) Thornton. It's the kind of game that I like to play."
|Marc Savard set to play in Game 7, no Bruins lineup changes
|05.14.09 at 12:01 pm ET
Bruins center Marc Savard isn't going to let a knee injury stop him from skating in Game 7 against the Carolina Hurricanes on Thursday night, and B's head coach Claude Julien said that his crafty pivot will be in the lineup. Savard suffered a knee-on-knee hit from Carolina winger Chad LaRose in the third period of Game 6, and exited the game early after Julien sent him to the dressing room.
Julien indicated on Thursday morning following the pre-game skate that the Bruins will skate the same lineup that took the ice in an impressive 4-2 victory over the Candy Canes on Tuesday night. That means Byron Bitz, who impressed with his blue collar work along the back wall with puck possession, will again skate with David Krejci and Michael Ryder.
"We'll have the same lineup tonight (as Game 6)," said Julien. "There shouldn't be any changes."
–Julien and his veteran players said that the most important key to success in the Game 7 setting is staying composed and keeping panic and chaos out of a game that can very easily spiral out of control.
The weight and pressure of season elimination can be a divisive influence that can pry a player out of their comfort zone and get a team out of their game plan — and Julien stressed it was important for his team to stick with the style of play that's resulted in two straight wins leading up to Thursday night.
"It's being composed. Being composed. You saw it in the game last night in Pittsburgh that they were able to come out and play their game and they were successful," said Julien. "You've got to be composed. I thought even last year in Montreal in Game 7 during the first period in Montreal, we were the better team.
"But sometimes you need the breaks and then (the Canadiens) got another goal in the second to make it 2-0. Had we stayed probably a little more composed, we could have battled back and got ourselves back into the game. But we were a young team last year. You hope what the young guys learned last year was to handle the pressure situation of Game 7 much better."
–B's center Stephane Yelle is playing in his 12th career Game 7 tonight against the Carolina Hurricanes, which is tops among all active NHL players and ranks him second in NHL history with Glenn Anderson and Ken Daneyko. The key to success from a guy that's been there nearly a dozen times:
"There'll be chatter before the game like there always is, and if the young guys have questions then they'll usually come and ask them," said Yelle. "Sticking with the game plan and not panicking (is key). When you start panicking you tend to get away from what you should be doing, and usually you feel like there's a ripple effect along the team and things turn to chaos. That's when you lose sight of what you're supposed to do.
"Different personalities will handle differently. I try to learn from my previous ones and go from there. My first year was Patty…Patty Roy and he had played in tons of them already. He was just a great leader and I followed him when I was younger being from Ottawa. I knew what he was all about. I just tried to watch him and see what he was doing (before Game 7)."
–Carolina winger Scott Walker has taken on the role of ultimate villian after sucker-punching Aaron Ward in the third period of Game 5 — and then subsequently skirting by with a simple $2500 slap of a fine — but said he's not going to get rattled by the "Ulf Treatment" at the Garden on Thursday night.
"I played in the minors a lot and it gets rowdy in some of those barns, and obviously in Vancouver it was a lot similar to this. A little bit in the New Jersey it rowdy behind the bench, but you don't really notice it that much. It's such an important game in Game 7 that I don't think the fans will affect the way they play or we play … or myself. It's great for them that they've got something to yell and scream about, but I don't think we'll lose our focus or anything like that."
–There seemed to be some indications out there that a potential Eastern Conference Finals Game 1 against the Pittsburgh Penguins would be scheduled at the TD Banknorth Garden on Sunday — likely Sunday afternoon. Stay tuned for that one because it could be another doubleheader if the Boston Celtics series goes to seven games.
|Savard and Kobasew bump the B's back up to 4-1
|05.12.09 at 8:03 pm ET
RALEIGH — 19:35 Booming one-timer from Staal in the left facoff circle off a pass from Ray Whitney, but Thomas came up with the juggling save.
17:11: The Bruins have been playing with fire for the last few minutes and they finally got burned. Shane Hnidy was beat to a puck in the corner by Matt Cullen, peeled off him and allowed him to get the net after he passed the puck and then Scott Walker found him in front. Thomas poked the puck away, but Cullen recovered and flipped a backhand past Thomas.
There was a scrum minute or two after the goal, and PJ Axelsson looked a little shaken up afterward. Keep an eye on that.
Things just turned up a notch physically. Erik Cole dumped Chara back behind the play with the Bruins moving the puck up the ice.
12:37: The Bruins have settled things down a little. A nice sequence there where Montador and Wideman were having trouble getting the puck out of the zone with speed, so Bergeron came back to help the B's move the puck swiftly through the neutral zone.
12:36: Great neutral zone pass by Byron Bitz to a rushing David Krejci, but the nifty centerman couldn't control the puck and shake his defender for a shot.
11″07: That was a big time play. Milan Lucic picked up the puck around the blue line and dangled through Tuomo Ruuto, Jussi Jokinen and Joni Pitkanen before dishing to a wide open Marc Savard at the right faceoff dot. Savard banged it into the open net, and the B's have answered the Canes challenge again.
9:00: Quick glove save on Scott Walker after a testing bid from the right faceoff dot.
5:49: Interference call on Pat Eaves when he dumped Axelsson in front of the Carolina net. First PP for the Bruins. The Cainiacs are sitting down with hands folded, and are pretty quiet right now.
5:30: Big time save by Thomas on Cullen during a short-handed rush when Wideman turned the puck over from the left point.
4:18: Big glove save by Ward on a Kessel snap shot on his trademark curl and drag move from the left side boards.
3:37: Wideman just hit a left post on a blast from the right faceoff circle after Kessel had rushed the puck up the ice. That was a full wind up and blast from Wideman that beat Ward cleanly.
1:57: Another score. Bergeron carried the puck down low in the Canes end and then fired a shot in front of the net just as Kobasew reached the post. Kobasew tucked the puck between Ward's pads for his third goal of the playoffs.
00:33: Mick Colageo of the New Bedford Times keeps calling Byron Bitz a "Young Man's Knuble" and I can't disagree. Bitz draws a penalty behind the net as Anton Babchuk basically hog-tied him to get the puck away from him. Somehow I don't see Blake Wheeler drawing a penalty like that behind the Boston net.
The Bruins are beating the Hurricanes by a 4-1 score after two full periods during Game 6 at the RBC Center in Raleigh, N.C.
|Bruins know 'how bad they want it'
|05.11.09 at 12:45 am ET
We want it as bad as you. It's been the slogan of these Boston Bruins all season.
On Sunday night, the Bruins showed their electrified fans how badly they want to become the first team in franchise history to come all the way back from a 3-1 series hole.
Sunday night's 4-0 shellacking of the Carolina Hurricanes was Step 1 in what they hope is a three-step journey to history.
"You can over systems as much as you want to but it really comes down to, 'How bad do you want it?' You've got to give Carolina credit," Bruins hitman Milan Lucic said. "They've just shown all series long that they've wanted it real bad and, especially Games 2,3 and 4, they really didn't give us nothing and were on us like crazy. Today we did a better job of keeping our composure and having that will to win." Read the rest of this entry »
|Don Cherry giving some big B's love on HNIC
|05.03.09 at 4:51 pm ET
Good Boston Bruins material from Don Cherry from 'Coach's Room' on Hockey Night in Canada over the last couple of weeks. Cherry has never shied away from showing some big, big love for his former team, and he fires off a few good rounds over the last few weeks of TV time.
The first CC is a walk down memory lane with the Lunchpail AC team he helped mold behind the Boston bench, and a comparison between B's goaltender Tim Thomas and Hall of Famer Gump Worsley given their similarly athletic, frenetic goaltending styles. There's no denying that some of the old school grainy Gump footage looks like a heck of a lot like Thomas when things get a little hectic around the Boston cage.
Here's another round of CBC's Coach's Corner from this weekend with Cherry continuing his long-standing hockey love affair with Marc Savard. Cherry also features what looks like a Bruins beanie baby on the HNIC desk while he and HNIC host Ron MacLean breeze their way through the intermission show. He starts it all by pronouncing "I'm on the (Bruins) bandwagon", and then lauds Savard for everything from his playmaking to the understated way he celebrates goals in Game 1 against the Hurricanes. Love the Scott Stevens "crazy eyes" shot as well.