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WO1999034820A1 - Nouvelle utilisation de la molecule de hb-gam - Google Patents

Nouvelle utilisation de la molecule de hb-gam Download PDF

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Publication number
WO1999034820A1
WO1999034820A1 PCT/FI1999/000009 FI9900009W WO9934820A1 WO 1999034820 A1 WO1999034820 A1 WO 1999034820A1 FI 9900009 W FI9900009 W FI 9900009W WO 9934820 A1 WO9934820 A1 WO 9934820A1
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Prior art keywords
gam
bone
medicament
carrier
expression
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PCT/FI1999/000009
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English (en)
Inventor
Heikki Matti Eemeli Rauvala
Shinji Imai
Marko Juhani Kaksonen
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Heikki Matti Eemeli Rauvala
Shinji Imai
Marko Juhani Kaksonen
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Application filed by Heikki Matti Eemeli Rauvala, Shinji Imai, Marko Juhani Kaksonen filed Critical Heikki Matti Eemeli Rauvala
Priority to AU19696/99A priority Critical patent/AU1969699A/en
Publication of WO1999034820A1 publication Critical patent/WO1999034820A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators

Definitions

  • This invention relates to bone fracture healing, stimulation of bone regeneration and repair.
  • the invention is specifically directed to an osteoblast specific molecule, HB-GAM, and to the use of this molecule for recruiting osteoblasts to a site of bone injury, such as a bone fracture, and stimulating bone formation.
  • the HB- GAM is applied locally at the site of bone injury.
  • the invention also provides a pharmaceutically acceptable composition for local use of HB-GAM, which is able to further accelerate the recovery of a bone injury.
  • Bone is a highly specialized form of connective tissue that has an extraordinary capacity for growth, continuous remodeling, and regeneration throughout life. Deposition of new bone by osteoblasts is strictly regulated to form appropriate shape and mass of the skeleton. It has been of great biological and clinical interest to identify molecular determinants of bone formation. Up to now, only few such molecular determinants have been proposed. For a current review on bone molecular biology, see Principles of Bone Biology (Eds. Bilezikian et al., Academic Press, 1996).
  • HB-GAM is a developmentally regulated extracellular matrix- associated protein originally identified from developing rat brain and shown to enhance neurite outgrowth of brain neurones.
  • HB-GAM is composed of 136 amino acids, rich in lysine and cysteine residues, and has a classic-type secretion signal.
  • the sequence of HB-GAM is conserved more than 95% in the human, rat, bovine and chicken and has 50% homology with midkine (MK) and retinoic acid-induced heparin-binding protein (R.IHB).
  • HB-GAM Osteoblast Specific Factor-1
  • OSF-1 Osteoblast Specific Factor-1
  • HB-GAM binds to osteoblast-type cell (Zhou, H. Y. et al., Biochem. Biophys. Res. Commun. 186, 1288-1293 (1992)) and enhances their spreading (Gieffers, C. et al., Eur. J. Cell Biol. 62, 352-361 (1993)) hint at a possible functional role of HB-GAM in bone tissue.
  • European patent EP-B 1-441763 relates to a placenta-derived heparin-binding growth factor, HBGF-8, and its corresponding human cDNA.
  • HB-GAM Speculations that HB-GAM could have a positive effect on bone regeneration, or resorption, have been mentioned in context with systemic diseases such as osteoporosis. However, the main emphasis has always been HB-GAM's well-established role as a brain mitogen.
  • the process of bone repair largely recapitulates a complexity of the embryonic bone formation.
  • the process consists of several stages; (1) induction of both chondrocytes and osteoblasts from undifferentiated mesenchymal cells, (2) formation of cartilaginous tissue by the induced chondrocytes within the bone defect, and (3) gradual resorption of the cartilage tissue and subsequent deposition of bone by the induced osteoblasts.
  • An artificial augmentation in any of these time-consuming stages would greatly facilitate fracture healing in man.
  • Surgical manipulation is today a major strategy in facilitating bone generation.
  • New clinical methods enhancing the rate and efficiency of fracture healing and promoting bone formation and healing in many other skeletal disorders are of great clinical and economic significance.
  • An effective medicament for enhancing and accelerating bone regeneration could be used in combination with the already existing surgical methods, thus increasing their success rate.
  • This medicament includes a therapeutically effective amount of HB-GAM in combination with a carrier.
  • the carrier is solid or moldable, and absorbable.
  • One preferred embodiment of the invention is to provide the medicament in the form of a synthetic bone graft, which can be inserted to the desired site surgically.
  • Another preferred embodiment of the invention is to provide the medicament in the form of solid particles or moldable matter, which can be inserted to the desired site by other means than open surgery, e.g. injection.
  • the medicament includes a solid, absorbable carrier.
  • the medicament is provided preferably in the form of artificial bone graft, or in the form of particles or moldable matter that can be inserted to the desired site without open surgery, e.g. by injection.
  • One preferred method of administration is to introduce the medicament to the site of injury in a surgical operation.
  • one preferred carrier is a synthetic bone graft.
  • Another preferred method of administration is to introduce the medicament to the site of injury without open surgery, e.g. by injection.
  • HB-GAM is upregulated in the cartilage matrix of fracture callus - in fact the repair of fracture recapitulates endochondral ossification of development.
  • the present invention applies recombinant HB-GAM with carrier materials that act as templates for endochondral ossification.
  • Experiment 5 shows that this accelerates bone healing substantially.
  • the most surprising effect of the present invention is indeed that the combination of HB-GAM with a suitable carrier can actually replace the time-consuming cartilage formation stage.
  • Example 5 clearly shows that osteoblasts are able to start bone tissue generation directly on the surface of the carrier containing HB-GAM.
  • Figure 1 Number of HB-GAM expressing osteocytes during repair of damaged bone in an arthritic rat model.
  • FIG. 1 Haptotaxis assay for osteoblast-like cells. Schematic presentation of the experimental design.
  • FIG. 1 Migration of osteblast-like cells vs. non-osteoblast cells. Open symbols: osteoblast-like cells; solid symbols: non-osteoblast-like cells.
  • FIG. 1 Schematic diagram of the gene construct used to produce transgenic mice.
  • FIG. 5 Expression of HB-GAM in transgenic mice, a) Western blot analysis of different neonatal tissues (postnatal day 1) from a transgene- positive mouse (lanes noted by +) and its transgene-negative littermate (noted by -). lanes b: brain, h: heart, m: femoral muscle, 1: liver; k: kidney. Note the most intense transgene expression in heart, b) Transgene expression in the periosteum in comparison to that found in the heart of 1 -year-old mice. Western blot analysis of tissues from two different transgenic lines (noted by +), and their non- transgenic littermates (noted by -).
  • HB-GAM-expression in the transgenic periostem is as high as or even higher than that in the transgenic heart (h).
  • lane c control recombinant HB-GAM protein (100 ng).
  • Figure 6. Histomorphometric analysis of femora of transgene-negative (Non tg) and transgene positive mice (Tg).
  • Figure 7 A suggested model of HB-GAM function in bone formation.
  • HB-GAM is a developmentally regulated extracellular matrix- associated protein suggested to play a role in development of the nervous system and the mesenchymal tissues.
  • the present invention shows that HB- GAM is highly expressed in the cartilage and bone matrices, onto which osteoblasts migrate, attach and deposit bone matrix during development.
  • cultured osteoblast-type cells migrate to matrix-bound HB-GAM in a transfilter assay.
  • the HB-GAM expression is downregulated after skeletal maturity, it is strongly upregulated in the adult periosteum during bone repair.
  • transgenic mice lines were produced that maintained a high expression level of HB-GAM in mesenchymal tissues until late adulthood.
  • the transgenic mice express HB-GAM in the adult bone at the level comparable to that observed in developing and regenerating bone. These mice develop a phenotype characterized by an increased amount of bone. These results demonstrate that HB-GAM plays an important role in regulating bone formation - probably by recruiting osteoblasts to the site of osteogenesis - and provide the basis for its clinical applications.
  • HB-GAM is an osteoblast-recruiting agent that is richly expressed in the cartilaginous tissue during development and repair of bone
  • implantation of HB-GAM in an artificial matrix can replace the stage of the cartilage formation.
  • the matrix is a synthetic bone graft or an absorbable, solid carrier.
  • the carrier is absorbable, elastic or moldable. Suitable materials for this purpose are described in e.g., international patent applications WO 92/00109, WO 96/03117 and WO 97/45367.
  • Such carriers may comprise e.g.
  • bioceramic systems bioactive glasses, silica-xerogels, collagen matrix, and the like, they are preferably resorbable, and they are pharmaceutically acceptable as carriers for administration of HB-GAM into the bone injury site.
  • Example 1 demonstrates that during development and growth of bone, HB-GAM is abundantly expressed at sites where osteoblasts attach and deposit bone matrix, i.e., (1) the cartilage matrix during endochondral ossification, (2) the epiphyseal growth plate, (3) the secondary ossification centers, and (4) the periosteum of growing bone.
  • matrix-bound HB-GAM recruits osteoblast-like cells (experiment 3). Taken together, HB-GAM is likely to play a critical role in bone formation by recruiting osteoblasts to the matrices onto which they deposit osteoid.
  • HB-GAM as a functional protein associated with bone formation further raise a question concerning its significance in relation to other molecular determinants of bone formation.
  • sequential gene expression alone produces only a rough model of a bone.
  • external influences are required so that the pre-existing bone can respond to refine its shape and mass. For instance, patterns of mechanical stress influence bone shapes like normal grooves, depressions, prominences and subtle curves.
  • Example 2 To date, there is no known molecular basis by which stress mediates new bone formation.
  • HB-GAM is upregulated in response to arthritic insult suggests a molecular model by which pre-existing bone may respond to external influence.
  • Stress sensor role of osteocyte has long been debated for its anatomical and cell biological characteristics. Osteocytes are dispersed throughout the mineralized matrix, and are connected with their neighbor osteocytes via long cell processes, which run through unmineralized matrix inside the canaliculi. The cell processes contact each other via gap junctions, thereby allowing cell-to-cell coupling.
  • Example 2 demonstrates that the number of HB-GAM-expressing osteocytes rapidly increases in response to arthritic insult to bone.
  • HB-GAM is secreted by osteocytes, penetrates through the bone matrix via canaliculi, and is deposited to the periosteum.
  • Figure 7 the inventors give a model for the role of HB-GAM in bone formation.
  • HB-GAM expression is enhanced in adjuvant-induced arthritis suggests that it may also be enhanced and cause undesired ossification in human diseases, e.g., bony ankylosis in rheumatoid arthritis, idiopathic spinal hyperostosis and ectopic ossification in damaged muscle (myositis ossificans).
  • HB-GAM might also have a possible role in the pathogenesis of different osteoconductive conditions. Inhibition of HB-GAM-mediated bone formation may become in future applicable to control undesired bone formation in human diseases.
  • Example 3 shows that the osteoblast-type cells migrate to and spread on HB-GAM in a manner that resembles the migratory response of neurons to matrix-bound HB-GAM.
  • the HB-GAM-induced migration of growth cone i.e., leading edge of neurons
  • N-syndecan a cell surface receptor of HB-GAM.
  • Osteoblasts in tissues and the osteoblast-type cells used in the present in vitro study express abundantly N-syndecan, which may thus mediate the migratory response in both osteoblasts and neurons.
  • HB-GAM expression in different tissues was carried out by Western blotting. Three mouse lines showing the highest expression level of the transgene were propagated for further analysis. A characteristic feature of these transgenic lines was the high expression level of HB-GAM in mesenchymal tissues (heart and muscle), whereas the littermates genotyped as negative control did not display any HB-GAM expression in the mesenchymal tissues analyzed shortly after birth. There was possibly some augmentation of HB-GAM expression in the brain of transgenic mice, but no expression in the liver or kidney.
  • a macroscopically detectable difference between the transgene- positive and transgene-negative mice was only found in bone (see below).
  • the HB-GAM expression in the periosteum was compared to the expression level found in the heart.
  • the HB-GAM expression in the periosteum of the transgene-positive individuals was as high as or even higher than the expression level found in heart. No expression could be detected either in the periosteum or in the heart of transgene-negative individuals of the same litter.
  • Estimated from the band intensities of Western blotting approximately 50 ⁇ g/g (wet tissue weight) of HB-GAM was found in the adult periosteum of the transgene-positive mice, which is comparable to the highest expression levels found in developing embryo.
  • HB-GAM was expressed by osteocytes dispersed in the cortical bone, and was localized along the surface of cortical bone in a similar manner to that found during the periosteal ossification. HB-GAM was also abundantly expressed in the epiphyseal growth plate, which was much wider than that of the non-transgenic littermates and was accompanied by more cancellous bone. Histomorphometric analysis showed that the cancellous bone volume, in addition to the cortical bone volume, was significantly larger in the transgene- positive mice. Another parameter, bone marrow area, a good indicator of osteoclast function, was not altered.
  • HB-GAM When HB-GAM is consistently produced under the beta-actin promoter, its expression level and pattern resembles that of regenerating bone in response to arthritic insult. The transgenic mice, over time, develop a phenotype marked by higher bone mass. This result, together with the strict regulation of endogeneous HB-GAM, suggests a coordinating role for HB- GAM in the regulation of physiological and pathological osteogenesis. Clinical application of this endogenously occurring, strictly regulated matrix- associated protein may provide novel approaches to control osteogenesis of man.
  • Examples 1-4 show HB-GAM to be an osteoblast-recruiting agent richly expressed in the cartilaginous tissue during development and repair of bone.
  • Experiment 5 was performed to show that the implantation of HB- GAM in an artificial matrix; e.g., agarose, can replace the stage of the cartilage formation and facilitate bone formation in fractures.
  • the formation of a bone bridge over the fracture site was accelerated from over 14 days to 10 days, which represents a 29% reduction in the time needed to reach this stage of recovery.
  • Such effect was quite unexpected and surprising, and illustrates well the improvement of bone reparation achieved by the present invention.
  • HB-GAM bone regeneration may be enhanced by surgical or otherwise local application of HB-GAM to guide and activate the deposition of new bone in a most favorable fashion.
  • the use of HB-GAM to enhance bone repair and bone fracture healing can be performed in connection to bone fractures treated by orthopedic surgery, in maxillofacial surgery, in treating bone defects following a surgical excision of bone affected by tumors.
  • the use of HB-GAM to enhance bone regeneration in connection with artificial prostheses surgery may also provide advantages.
  • Another preferred embodiment of the invention is administering HB-GAM in a suitable carrier to a fracture site or a site of bone injury without the use of open surgery.
  • Recombinant HB-GAM was produced with the aid of a baculovirus vector and purified to apparent homogeneity as described by Raulo, E.et al.in J. Biol. Chem. 267, 11408-11416 (1992).
  • Antibodies to the purified recombinant HB-GAM were produced in rabbit and affinity-purified.
  • the antibodies have been characterized using Western blotting and their specificity against HB-GAM has been verified in an immunohistochemical context (Peng, H.B. et al., J. Neurosci. 15, 3027- 3038 (1995)).
  • Immuno-electron microscopy Immuno-electron microscopy. Immunostained sections for electron microscopy were postfixed with 1 % osmium tetroxide for 1 h and dehydrated in ethanol series. The sections were flat embedded in epon medium and coverslipped on glass slides coated with silicon. Light-microscopic observation was made at this point in order to select areas to be examined electron-microscopically. Ultra-thin sections (60 nm) were stained with uranyl acetate and lead citrate, and were examined by a transmission electron microscope (Acceleration voltage: 60 KeV: Joel 1200, Joel, Tokyo, Japan).
  • Rat osteogenic sarcoma cell line UMR- 106 (American Type Culture Collection (ATCC) CRL-1661), and human osteogenic sarcoma cell lines, Saos-2 (ATCC HTB-85), U-2 OS (ATCC HTB- 96), and KHOS/NP (ATCC CRL-1544) were freshly prepared in the medium recommended by the ATCC, supplemented with 10% letal calf serum (FCS), 100 U/ml penicillin G and 100 ⁇ g/ml streptomycin.
  • FCS letal calf serum
  • 3T3 mouse fibroblasts and N18 mouse neuroblastoma cells were used as control cells.
  • HB-GAM The expression pattern of HB-GAM was studied in rat and the expression was detected by in situ hybridization and immuno-electron microscopy.
  • HB- GAM HB- GAM
  • HB-GAM expression persisted in the cartilage matrix during endochondral ossification on embryonic day 20.
  • In situ hybridization and immuno-electron microscopy showed that chondrocytes synthesized HB- GAM and localized it to the chondrocytes in the cartilage matrix.
  • the sense probe used as a control did not give any signal.
  • Postnatal HB- GAM expression persisted in the epiphyseal growth plate of the humeral head (postnatal day 7).
  • transient expression was observed in the secondary ossification centers appearing shortly after birth and in the periosteum of cortical bone. Control sections incubated with non-specific rabbit IgG did not give any staining.
  • Freund's complete adjuvant cell wall fragments of heat-killed mycobacterium butylicum ;
  • HB-GAM expression was limited, and only few osteocytes expressed HB-GAM. After adjuvant treatment the number of HB-GAM-expressing osteocytes increased on day 7, and was maintained high until day 21. HB-GAM was distributed on the cell surface of osteocytes, but more prominently on the walls of both osteocytic lacunae and canaliculi.
  • Osteocytes are connected with bone surface via their cell processes that run through the canaliculi.
  • HB-GAM appeared to be localized to the bone surface via the osteocytic processes. Accordingly, the periosteum became strongly immunoreactive to HB-GAM on day 10. The periosteum proliferates in response to inflammatory insult and the thickened periosteum was rich in HB-GAM.
  • HB-GAM-expressing osteocytes in the cortical bone adjacent to the periosteal ossification are shown in Figure 1.
  • HB-GAM-expressing osteocytes were calculated from a square (0.2mm x 0.2mm) on electron microscopy sections. Three sections were counted for each animal (3 animals/time point), and the bars represent mean + s.e.m.
  • HB-GAM recombinant HB-GAM was coated to the lower surface of the filter at doubling concentrations from 0.025 ⁇ g/ml to 50.0 ⁇ g/ml overnight at 4 °C. The filter was then thoroughly washed with water, and dried in laminary flow hood. The chamber was filled with the FCS-free medium containing 1 mg/ml bovine serum albumin (BSA) 30 min before adding cells. Cells were placed in the upper chamber at approximately 1.0 x 10 5 cells/cm 2 , and cultured for 4 h.
  • BSA bovine serum albumin
  • Osteoblast-like cells (rat osteogenic sarcoma cell line UMR-106, and human osteogenic sarcoma cell lines Saos-2, U-2 and KHOS/NP) migrated through the pores, and then spread on the HB-GAM-coated surface of the filter. Less than 1% of plated UMR-106 cells migrated to the control matrices (non specific rat IgG, 0.92%; bovine serum albumin, 0.89%). All the osteoblast- like cells migrated in a dose-dependent manner, but non-osteoblastic cells, 3T3 fibroblasts and N18 neuroblastoma cells, did not respond to HB-GAM as shown in Figure 3.
  • HB-GAM stimulates bone formation was tested by producing transgenic mice that maintain a high expression level of osseous HB-GAM until adulthood. Over time, the cortical and cancellous bone mass is significantly increased in the transgenic mice. The thickness of the cortical bone is increased up to 147.4% by 1-year of age. The increase in bone mass is likely to result from an augmented osteoblast-recruiting activity, that is maintained until late adulthood.
  • HB-GAM cDNA was cloned into a BamHI restriction site in pHBApr-1-neo vector.
  • pHBApr-1-neo contains the promoter and the first intron from human beta-actin gene and SV40- polyadenylation signal (Gunning, P.et al., Proc. Natl. Acad. Sci. 84, 4831- 4835 (1987)).
  • the expression casette was removed from the plasmid by digestion with EcoRI and Ndel, separated by agarose gel electrophoresis, and purified. Fertilized eggs from FVB/NIH and FlxNMRI mice were used for microinjection. Founder animals were screened by Southern blotting from tail biopsies. Founder animals were bred with FVB/NIH mice.
  • Histopathologic and histomorphometric analysis Gross appearance of the skeletons was evaluated after maceration in potassium hydroxide and removal of the soft tissue. Topographical expression patterns of HB-GAM were studied using longitudinal and cross-sectional sections of femora and humeri of 1 -year-old animals (2 transgenic and 2 non-transgenic animals). Histomorphometric analyses, following the procedures and nomenclature recommended in Parfitt, A.M. et al. Bone histomorphometry: Standardization of nomenclature, symbols, and units. J. Bone Miner. Res.
  • the femora of the transgene positive mouse had an ivory-like solid appearance, whereas the transgene-negative femur displayed a brownish surface due to the bone marrow structures seen through the cortical bone. Brownish marrow structure was also seen in the transgene-negative ulna. The ivory-like appearance was particularly prominent in the scapular spinus of the transgene-positive mouse. The transparency of scapular plane in the transgene-negative bone was notable.
  • Table 1 Sizes of the femur and humerus from transgenic and non-transgenic mice. Thickness describes thickness of the cortical bone. Parameters for diaphysis are measured at the midpoint of longitudinal lenght, and those for metaphysis are measured at quarter of the longitudinal length apart from either humeral or femoral head. Sizes are in micrometers (mean + s.e.m).
  • Diaphysis [ Diameter Thickness 196 ⁇ 27 289 ⁇ 40 0.0005
  • a bone defect was made in mid-frontal aspect of the tibia of 7-week old Wistar Lewis Rats using a dental drill (diameter: 3mm).
  • Agarose was initially melt in distilled water at boiling temperature (10% w/v), and was gradually cooled down to 37 °C.
  • Recombinant HB-GAM (10 ⁇ g/ml) was mixed to the agarose, and further cooled down to 4 °C in order to solidify. Cubic blocks of the HB-GAM-agarose were excised (3mm x 3mm x 1mm), and placed in the bone defect. The subsequent histological and radiographic changes were compared to those of negative control [HB-GAM (-), agarose (+)] and normal control [HB-GAM (-), agarose (-)].
  • Radiographic calcification around the HB-GAM-agarose was detected as early as 5 days after implantation (day 5), whereas it was never observed in negative control or normal control animals. Histological examination disclosed thin, egg shell-like bone formation around the HB-GAM -agarose blocks, whereas the negative control blocks accompanied a mild, foreign body reaction (day 7). By radiographic examination, the calcification around the HB-GAM-agarose blocks rapidly grew, and the bone defect was already bridged at day 10. In contrast, the bridging of the defects took more than 14 days in negative and normal control. However, histological examination on samples from the later days disclosed that the HB-GAM agarose blocks were not replaced by bone, and remained within the newly induced bone tissue.
  • HB-GAM-agarose to the bone defect indeed replaced the process of cartilage tissue formation, and recruited the induced osteoblasts. Consequently, the initial process of the bone formation is greatly facilitated.
  • the physiological repair process also requires a concomitant resorption of the cartilage tissue. Since the agarose matrix used in this study is not absorbable in a manner similar to that of the cartilage tissue, the newly induced bone did not replace the block.
  • the use of other, biocompatible and absorbable materials as a carrier of HB-GAM may thus provide a better result by enabling subsequent introduction of blood vessels and bone marrow formation.

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Abstract

L'invention porte sur un médicament spécifique de l'os à usage local accélérant et renforçant la régénération de l'os suite à des fractures et autres dommages. Ledit médicament comporte une dose à effet thérapeutique d'HB-GAM (molécule de croissance des fibroblastes fixant l'héparine) combinée à un excipient, qui, dans l'une des exécutions préférées, est solide et résorbable. Dans une autre exécution préférée, le médicament se présente sous la forme d'une greffe osseuse artificielle s'implantant chirurgicalement dans le site désiré. Dans une autre exécution préférée, le médicament se présente sous la forme de particules solides s'implantant dans le site désiré par des moyens non chirurgicaux, par exemple par injection.
PCT/FI1999/000009 1998-01-09 1999-01-08 Nouvelle utilisation de la molecule de hb-gam WO1999034820A1 (fr)

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FI980032A FI980032A0 (fi) 1998-01-09 1998-01-09 Ny anvaendning av en heparin bindande, med tillvaext foerknippad molekyl

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Cited By (1)

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Publication number Priority date Publication date Assignee Title
EP1084717A1 (fr) * 1999-09-17 2001-03-21 Depuy Orthopaedics, Inc. Compositions à base de Pléiotrophine activant la réparation du tissu conjonctif

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WO1996003117A1 (fr) * 1994-07-27 1996-02-08 The Trustees Of The University Of Pennsylvania Incorporation de molecules biologiquement actives dans des verres bioactifs
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WO1992000324A1 (fr) * 1990-06-29 1992-01-09 Hoechst Japan Limited Nouvelle proteine apte a provoquer une croissance cellulaire differentielle, et son procede de production employant la technologie du genie genetique
WO1996003117A1 (fr) * 1994-07-27 1996-02-08 The Trustees Of The University Of Pennsylvania Incorporation de molecules biologiquement actives dans des verres bioactifs
WO1997045367A1 (fr) * 1996-05-29 1997-12-04 Orion-Yhtymä Oy Oxydes solubles pour applications biologiques

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MASUDA H ET AL: "Bone loss due to estrogen deficieny is compensated in transgenic mice overexpressing human osteoblast stimulating factor-1", BIOCHEM. BIOPHYS. RES. COMMUN., vol. 238, 1997, pages 528 - 533, XP002100860 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1084717A1 (fr) * 1999-09-17 2001-03-21 Depuy Orthopaedics, Inc. Compositions à base de Pléiotrophine activant la réparation du tissu conjonctif
US6364912B1 (en) 1999-09-17 2002-04-02 Depuy Orthopeaedics, Inc. Pleiotrophin-based compositions for enhancing connective tissue repair

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