Suche Bilder Maps Play YouTube News Gmail Drive Mehr »
Anmelden
Nutzer von Screenreadern: Klicke auf diesen Link, um die Bedienungshilfen zu aktivieren. Dieser Modus bietet die gleichen Grundfunktionen, funktioniert aber besser mit deinem Reader.

Patentsuche

  1. Erweiterte Patentsuche
VeröffentlichungsnummerUS20060216325 A1
PublikationstypAnmeldung
AnmeldenummerUS 11/433,968
Veröffentlichungsdatum28. Sept. 2006
Eingetragen15. Mai 2006
Prioritätsdatum14. Aug. 1997
Auch veröffentlicht unterCA2300415A1, CA2300415C, DE69714035D1, DE69714035T2, EP0896825A1, EP0896825B1, US6514514, US6582471, US20030236574, US20090297605, USRE41286, WO1999008728A1
Veröffentlichungsnummer11433968, 433968, US 2006/0216325 A1, US 2006/216325 A1, US 20060216325 A1, US 20060216325A1, US 2006216325 A1, US 2006216325A1, US-A1-20060216325, US-A1-2006216325, US2006/0216325A1, US2006/216325A1, US20060216325 A1, US20060216325A1, US2006216325 A1, US2006216325A1
ErfinderBrent Atkinson, Pedro Bittman, James Benedict, John Ranieri, Marsha Whitney, Donald Chickering
Ursprünglich BevollmächtigterAtkinson Brent L, Pedro Bittman, Benedict James J, John Ranieri, Whitney Marsha L, Donald Chickering
Zitat exportierenBiBTeX, EndNote, RefMan
Externe Links: USPTO, USPTO-Zuordnung, Espacenet
Composition and device for in vivo cartilagerepair
US 20060216325 A1
Zusammenfassung
The composition as described serves for in vivo cartilage repair. It basically consists of a naturally derived osteoinductive and/or chondroinductive mixture of factors (e.g. derived from bone) or of a synthetic mimic of such a mixture combined with a nanosphere delivery system. A preferred mixture of factors is the combination of factors isolated from bone, known as BP and described by Poser and Benedict (WO 95/13767). The nanosphere delivery system consists of nanospheres defined as polymer particles of less than 1000 nm in diameter (whereby the majority of particles preferably ranges between 200-400 nm) in which nanospheres the combination of factors is encapsulated. The nanospheres are loaded with the mixture of factors in a weight ratio of 0.001 to 17% (w/w), preferably of 1 to 4% (w/w) and have a release profile with an initial burst of 10 to 20% of the total load over the first 24 hours and a long time release of at least 0.1 per day during at least seven following days. The nanospheres are composed of e.g. ((D,L)-lactic acid/glycolic acid)-copolymer (PLGA). The loaded nanospheres are e.g. made by phase inversion. The composition is advantageously utilized as a device comprising any biodegradable matrix in which the nanospheres loaded with the factor combination is contained.
Bilder(6)
Previous page
Next page
Ansprüche(21)
1-23. (canceled)
24. A composition comprising:
a chondroinductive protein mixture; and
a delivery system having an initial first release rate (% total protein mixture load/time) greater than a subsequent second release rate (% total protein mixture load/time).
25. The composition of claim 1, wherein the first rate is greater than 10% per day.
26. The composition of claim 1, wherein the second rate is less than 1% per day.
27. The composition of claim 1, wherein the delivery system has a third release rate.
28. The composition of claim 4, wherein the third release rate is greater than 0.1% per day.
29. The composition of claim 4, wherein the third release rate occurs temporally before the second release rate.
30. The composition of claim 1, wherein the protein mixture comprises a bone derived protein.
31. The composition of claim 1, wherein the delivery system comprises nanospheres.
32. The composition of claim 1, further comprising a matrix.
33. The composition of claim 9, wherein the matrix comprises collagen.
34. A composition comprising:
a chondroinductive protein mixture; and
a delivery system having an initial first release rate greater than 10% total protein mixture load per day and a subsequent second release rate less than 1% total protein mixture load per day.
35. The composition of claim 10, wherein the delivery system has a third release rate.
36. The composition of claim 12, wherein the third release rate is greater than 0.1% per day.
37. The composition of claim 12, wherein the third release rate occurs temporally before the second release rate.
38. The composition of claim 10, wherein the protein mixture comprises a bone derived protein.
39. The composition of claim 10, wherein the delivery system comprises nanospheres.
40. The composition of claim 10, further comprising a matrix.
41. The composition of claim 17, wherein the matrix comprises a material selected from the group consisting of type I collagen, type II collagen and hyaluronic acid.
42. The composition of claim 17, wherein the matrix comprises a material selected from the group consisting of type I collagen.
43. A composition comprising:
a bone derived chondroinductive protein mixture;
a biodegradable matrix including collagen; and
a nanosphere delivery system having an initial first release rate greater than 10% total protein mixture load per day and a subsequent second release rate less than 1% total protein mixture load per day.
Beschreibung
    BACKGROUND OF THE INVENTION
  • [0001]
    Articular cartilage, an avascular tissue found at the ends of articulating bones, has no natural capacity to heal. During normal cartilage ontogeny, mesenchymal stem cells condense to form areas of high density and proceed through a series of developmental stages that ends in the mature chondrocyte. The final hyaline cartilage tissue contains only chondrocytes that are surrounded by a matrix composed of type II collagen, sulfated proteoglycans, and additional proteins. The matrix is heterogenous in structure and consists of three morphologically distinct zones: superficial, intermediate, and deep. Zones differ among collagen and proteoglycan distribution, calcification, orientation of collagen fibrils, and the positioning and alignment of chondrocytes (Archer et al., J. Anat. 189(1): 23-35, 1996; Morrison et al., J. Anat. 189(1): 9-22 1996, Mow et al., Biomaterials 13(2): 67-97, 1992). These properties provide the unique mechanical and physical parameters to hyaline cartilage tissue.
  • [0002]
    In 1965, a demineralized extraction from bovine long bones was found to induce endochondral bone formation in the rat subcutaneous assay (Urist Science 150: 893-899, 1965). Seven individual factors, termed Bone Morphogenetic Proteins (BMPs), were isolated to homogeneity and, because of significant sequence homology, classified as members of the TGFβ super-family of proteins (Wozney, et al., Science 242: 1528-34, 1988; Wang et al., Proc. Nat. Acad. Sci. 87: 2220-2224, 1990). These individual, recombinantly-produced factors also induce ectopic bone formation in the rat model (Luyten et al., J. Biol. Chem. 264: 13377-80, 1989; Celeste et al., Proc. Nat. Acad. Sci. 87: 9843-50, 1990). In addition, in vitro tests have demonstrated that both BMP-2 and TGFβ-1 induce mesenchymal stem cells to form cartilage (Denker, et al., Differentiation 59(1): 25-34, 1995; Denker et al., 41st Ann. Orthop. Res. Society 465: 1995). Both BMP-7 and BMP-2 have been shown to enhance matrix production of chondrocytes in vitro (Flechtenmacher J. Arthritis Rheum. 39(11): 1896-904, 1996: Sailor et al., J. Orthop. Res. 14: 937-945, 1996). From these data we can conclude that not only are the BMPs important regulators of osteogenesis, but that they also play crucial roles during chondrogenic development in vitro.
  • [0003]
    A partially-purified protein mixture from bovine long bones, termed BP (Bone Protein), also induces cartilage and bone formation in the rat subcutaneous assay (Poser and Benedict, WO95/13767). BP in combination with calcium carbonate promotes bone formation in the body. In vitro, BP induces mesenchymal stem cells to differentiate specifically to the cartilage lineage, in high yields, and to late stages of maturation (Atkinson et al., J. Cellular Biochem. 65: 325-339, 1997).
  • [0004]
    The molecular mechanism for cartilage and bone formation has been partially elucidated. Both BMP and TGFβ molecules bind to cell surface receptors (the BMP/TGFβ receptors), which initiates a cascade of signals to the nucleus that promotes proliferation, differentiation to cartilage, and/or differentiation to bone (Massague Cell 85: 947-950, 1996).
  • [0005]
    In 1984, Urist described a substantially pure, but not recombinant BMP, combined with a biodegradable polylactic acid polymer delivery system for bone repair (U.S. Pat. No. -4,563,489). This system blends together equal quantities of BMP and polylactic acid (PLA) powder (100 μg of each) and decreases the amount of BMP required to promote bone repair.
  • [0006]
    Hunziker (U.S. Pat. No. -5,368,858; U.S. Pat. No. -5,206,023) describes a cartilage repair composition consisting of a biodegradable matrix, a proliferation and/or chemotactic agent, and a transforming factor. A two stage approach is used where each component has a specific function over time. First, a specific concentration of proliferation/chemotactic agent fills the defect with repair cells. Secondly, a larger transforming factor concentration transforms repair cells into chondrocytes. Thereby the proliferation agent and the transforming agent may both be TGFβ differing in concentration only. In addition, the patent discloses a liposome encapsulation method for delivering TFGβ-1 serving as transformation agent.
  • [0007]
    Hattersley et al. (WO 96/39170) disclose a two factor composition for inducing cartilaginous tissue formation using a cartilage formation-inducing protein and a cartilage maintenance inducing protein. Specific recombinant cartilage formation inducing protein(s) are specified as BMP-13, MP-52, and BMP-12, and cartilage maintenance-inducing protein(s) are specified as BMP-9. In one embodiment, BMP-9 is encapsulated in a resorbable polymer system and delivered to coincide with the presence of cartilage formation inducing protein(s).
  • [0008]
    Laurencin et al., (U.S. Pat. No. -5,629,009) disclose a chondrogenesis-inducing device, consisting of a polyanhydride and polyorthoester, that delivers water soluble proteins derived from demineralized bone matrix, TGFβ, EGF, FGF, or PDGF.
  • [0009]
    The results of the approaches to cartilage repair as cited above are encouraging but they are not satisfactory. In particular, the repair tissue arrived at is not fully hyaline in appearance and/or it does not contain the proper chondrocyte organization. Furthermore, previous approaches to cartilage repair have been addressed to very small defects and have not been able to solve problems associated with repair of large, clinically relevant defects.
  • [0010]
    One reason that previous approaches failed to adequately repair cartilage may be that they were not able to recapitulate natural cartilage ontogeny faithfully enough, this natural ontogeny being based on a very complicated system of different factors, factor combinations and factor concentrations with temporal and local gradients. A single recombinant growth factor or two recombinant growth factors may lack the inductive complexity to mimic cartilage development to a sufficient degree and/or the delivery systems used may not have been able to mimic the gradient complexity of the natural system to a satisfactory degree.
  • [0011]
    Previous approaches may also have failed because growth factor concentrations were not able to be maintained over a sufficient amount of time, which would prevent a full and permanent differentiation of precursor cells to chondrocytes. The loss of growth factor could be caused by diffusion, degradation, or by cellular internalization that bypasses the BMP/TGFβ receptors. Maintaining a sufficient growth factor concentration becomes particularly important in repair of large sized defects that may take several days or several weeks to fully repopulate with cells.
  • [0012]
    The object of this invention is to create a composition for improved cartilage repair in vivo. The inventive composition is to enable in vivo formation of repair cartilage tissue which tissue resembles endogenous cartilage (in the case of articular cartilage with its specific chondrocyte spatial organization and superficial, intermediate, and deep cartilage zones) more closely than repair tissue achieved using known compositions for inducing cartilage repair. A further object of the invention is to create a device for cartilage repair which device contains the inventive composition.
  • [0013]
    This object is achieved by the composition and the device as defined by the claims.
  • BRIEF DESCRIPTION OF THE INVENTION
  • [0014]
    The inventive composition basically consists of a naturally derived osteo-inductive and/or chondroinductive mixture of factors (e.g. derived from bone) or of a synthetic mimic of such a mixture combined with a nanosphere delivery system. A preferred mixture of factors is the combination of factors isolated from bone, known as BP and described by Poser and Benedict (WO 95/13767). The nanosphere delivery system consists of nanospheres defined as polymer particles of less than 1000 nm in diameter (whereby the majority of particles preferably ranges between 200-400 nm) in which nanospheres the combination of factors is encapsulated. The nanospheres are loaded with the mixture of factors in a weight ratio of 0.001 to 17% (w/w), preferably of 1 to 4% (w/w) and have an analytically defined release profile (see description regarding FIG. 2) showing an initial burst of 10 to 20% of the total load over the first 24 hours and a long time release of at least 0.1 per day during at least seven following days, preferably of 0.1 to 1% over the following 40 to 60 days. The nanospheres are composed of e.g. (lactic acid-glycolic acid)-copolymers (Poly-(D,L)lactic acid-glycolic acid) made of 20 to 80% lactic acid and 80 to 20% of glycolic acid, more preferably of 50% lactic acid and 50% of glycolic acid.
  • [0015]
    The loaded nanospheres are e.g. made by phase inversion according to Mathiowitz et al. (Nature, 386: 410-413, 1997) or by other methods known to those skilled in the art (Landry, Ph.D Thesis, Frankfurt, Germany).
  • [0016]
    The inventive composition is advantageously utilized as a device comprising any biodegradable matrix including collagen type I and II, and hyaluronic acid in which matrix the nanospheres loaded with the factor combination is contained. The matrix can be in the form of a sponge, membrane, film or gel. The matrix should be easily digestible by migrating cells, should be of a porous nature to enhance cell migration, and/or should be able to completely fill the defect area without any gaps.
  • [0017]
    It is surprisingly found that the inventive composition consisting of an osteo-inductive and/or chondroinductive combination of factors (e.g. derived from natural tissue) encapsulated in nanospheres as specified above, if applied to a defect area of an articular cartilage, leads to the transformation of virtually all precursor cells recruited to the repair area to chondrocytes, and furthermore, leads to a homogenous chondrocyte population of the repair area and to a chondrocyte order and anisotropic appearance as observed in endogenous hyaline cartilage. These findings encourage the prospect that the inventive composition may lead to significant improvements also regarding repair of large defects.
  • [0018]
    As mentioned above, instead of an osteoinductive and/or chondroinductive mixture of factors derived from bone (BP), the inventive composition may comprise natural factor mixtures derived from other tissues (e.g. cartilage, tendon, meniscus or ligament) or may even be a synthetic mimic of such a mixture having an osteoinductive and/or chondroinductive effect. Effective mixtures isolated from natural tissue seem to contain a combination of proliferation, differentiation, and spatial organizing proteins which in combination enhance the tissue rebuilding capacity more effectively than single proteins (e.g. recombinant proteins).
  • [0019]
    The specified, analytically defined release profile of such factor mixtures from nanospheres results in the formation of concentration gradients of proliferation and differentiation factors, which obviously mimics the complex gradients of factors observed during natural development very well. The nanosphere extended release profile is sufficient to provide growth factor during the time frame that repair cells arrive into the matrix. The release profile obviously leads to a homogenous population of a matrix with precursor cells, to full differentiation of virtually all of the precursor cells to chondrocytes, and to the formation of an endogenous hyaline cartilage structure.
  • [0020]
    Another advantage of the inventive composition is that when the nanospheres are placed in a matrix to form a device for cartilage repair, they are randomly distributed and remain in place when in a joint cartilage defect. During cellular infiltration and differentiation, the nanospheres are in the correct position over the correct time frame.
  • [0021]
    Nanospheres have been demonstrated to adhere to the gastrointestinal mucus and cellular linings after oral ingestion (Mathiowitz et al., Nature, 386 410-413 1997). We envisage that nanospheres also adhere to cartilage precursor cells and furthermore, may also adhere to BMP/TGFβ receptors located on the cell membrane. This property allows localized high-efficiency delivery to the target cells and/or receptors. Because of the nanosphere small size and the chemical properties, they are more effective than liposomes or diffusion controlled delivery systems. The efficient delivery to the receptors will facilitate chondrogenesis.
  • [0022]
    Derived from the above findings, we envisage the following mechanism for cartilage repair using the effect of the inventive composition. During the first 24 hours (initial burst) 10 to 20% of the total load of the factor mixture is released from the nanospheres into the matrix and diffuses into the synovial environment. Following the initial burst, the nanospheres begin to release factors at a slow rate, which produces gradients of proliferation, differentiation, and spatial organizing proteins. In response to such gradients, precursor cells migrate to the defect site. The loaded nanospheres adhere to cartilage precursor cells and to the BMP and TGFβ receptors to provide localized highly efficient delivery. The precursor cells become differentiated to chondrocytes and secrete type II collagen and cartilage-specific proteoglycans. The composition of the present invention stimulates differentiation of virtually all of these cells to overt chondrocytes and induces an ordered cartilage structure which closely resembles hyaline cartilage. Furthermore, we envisage that this release system will allow homogenous repair of large defect sites and repair of defects from patients with low quantities of precursor cells.
  • [0023]
    For in vivo cartilage repair, the inventive device consisting of a matrix and the loaded nanospheres is placed in a chondral lesion that was caused by trauma, arthritis, congenital, or other origin. The damage can result in holes or crevices or can consist of soft, dying, or sick cartilage tissue that is removed surgically prior to implantation of the device. Because of the unique properties of the inventive device precursor cells populate the matrix, differentiate to chondrocytes, and form hyaline cartilage.
  • [0024]
    Application of the inventive composition (without matrix) e.g. by injection can be envisaged also, in particular in the case of small defects. Thereby at least 2 μg of the composition per ml of defect size is applied or at least 20 ng of the osteoinductive and/or chondroinductive mixture encapsulated in the nanospheres is applied per ml defect size.
  • [0025]
    The inventive composition and the inventive device are suitable for repair of cartilage tissue in general, in particular for articular cartilage and for meniscus cartilage.
  • BRIEF DESCRIPTION OF THE FIGURES
  • [0026]
    The following figures illustrate the physical and chemical parameters of the inventive composition, the in vitro cartilage inductive activity of BP released from nanospheres and in vivo repair of an articular cartilage defect using the inventive device.
  • [0027]
    FIG. 1 shows a scanning electron micrograph of BP-loaded nanospheres;
  • [0028]
    FIG. 2 shows the release profile (cumulative release vs. time) of the inventive composition;
  • [0029]
    FIG. 3 shows the release profile of the inventive composition compared with release profiles of nanosphere delivery systems loaded with other proteins;
  • [0030]
    FIG. 4 shows the volume of a cartilage defect vs. the days required for populating the defect with repair cells;
  • [0031]
    FIG. 5 shows micromass cultures in the presence or absence of nanospheres loaded with BP;
  • [0032]
    FIG. 6 shows cartilage marker analyses for in vitro cultures containing BP only and for similar cultures containing the inventive composition;
  • DETAILED DESCRIPTION OF THE INVENTION
  • [0033]
    FIG. 1 shows a scanning electron micrograph of BP-loaded nanospheres. The microparticle sizes range from 100-1000 nm with the majority of individual particles ranging between 200-400 nm.
  • [0034]
    The release rate profile of the inventive composition was determined by in vitro analysis of BP delivered from nanospheres. These nanospheres were made by phase inversion according to the method as disclosed by Mathiowitz et al. (Nature 386, 410-414, 1997) of ((DL)lactic acid/glycolic acid)-copolymer containing the two acids in a weight ratio of 50:50 and they were loaded with 1% and with 4% of BP.
  • [0035]
    For determination of the release rate profile, the nanospheres were placed in a sterile saline solution and incubated at 37° C. BP released into the supernatant was measured using a BCA assay (Pierce). BP released from the nanospheres as specified shows two successive and distinct profile parts: a fast release (initial burst) of approximately 10 to 20% of the loaded BP over the first 24 hours and a slow release of 0.1 to 1% per day (cumulative 40% to 50%) over 40 to 60 days (FIG. 2).
  • [0036]
    The release is intermediate between zero-order and first-order kinetics. Both the 1% and 4% encapsulated BP have similar release profiles.
  • [0037]
    For attaining release rate profiles as specified above and as necessary for the improved results in cartilage repair the nanospheres are to be adapted accordingly when using factor mixtures other than BP. Thereby, e.g the composition of the nanosphere copolymer, the molecular weight of the polymer molecules and/or the loading percentage of the nanospheres may be changed. The optimum nanosphere character for each specific case has to be found experimentally whereby the release rate profile is analyzed in vitro as described above.
  • [0038]
    In the same way, the nanosphere delivery system can be modified regarding the percentage of BP to be released in the first 24 hours, percentage of BP to be released after 24 hours and/or length of time after the first 24 hours during which the remainder of BP is released. In addition, the percentage of BP loaded to the nanospheres is of course variable too, whereby for obtaining the results as described for the specified composition. all the modifications are to be chosen such that the resulting delivery keeps within the range as specified.
  • [0039]
    All of the above parameters can be modified to account for the patient's age, sex, diet, defect location, amount of blood present in the defect, and other clinical factors to provide optimal cartilage repair. For example, nanospheres with longer release rates are used for treating larger defects and/or for patients with fewer precursor cells (e.g. older patients or patients with degenerative symptoms). In contrast, patients with larger quantities of progenitor cells and/or smaller defects may require a shorter release rate profile.
  • [0040]
    FIG. 3 shows the release profile as shown in FIG. 2 for nanospheres as specified above loaded with BP and with other proteins (same loading percentages) such as BSA (bovine serum albumin) or lysozyme. The drastically different release characteristics shows that the profile is dependent on the protein type also. Tne same is valid for a more hydrophobic mixture of bovine bone derived proteins (PIBP).
  • [0041]
    FIG. 3 illustrates the singularity of the inventive combination consisting of the specific delivery system (nanospheres as specified above encapsulating the factors) and the specific protein mixture (BP) which is obviously the key to the improved results in cartilage repair as observed when using the inventive composition or device.
  • [0042]
    To determine the length of time required for precursor cell repopulation of different sized defects, the following calculation was performed. We estimate that approximately 50,000 cells are recruited to the defect/day. Since the cellular density of cartilage is about 4×107 cells/ml, a 10 μl volume defect will take approximately 8 days to fill with cells. FIG. 4 plots the number of days required to fill different volume defects with cells. The Figure assumes an infinite supply of cells and a constant rate of cell attraction to the defect site, The graph demonstrates that the larger a defect size is, the more time is required to completely fill it with cells. Since a 60 μl volume defect will take over 45 days to fill, this Figure demonstrates the necessity for a long term release of factors to induce differentiation of the precursor cells over up to a two month period.
  • [0043]
    To determine whether BP bioactivity is harmed by the encapsulation process and to determine whether the released BP was fully bioactive, the following assay was performed. Previously, it was demonstrated that 10T1/2 micromass cultures exposed to BP induce formation of a three dimensional spheroid structure that can be observed macroscopically in tissue culture wells (Atkinson et al., J. Cellular Biochem. 65: 325-339, 1997). BP concentrations equal or greater than 20 ng/ml were required for spheroid formation. No spheroid forms in the absence of BP or at concentrations less than 10 ng/ml (see following table). In this assay, 10T1/2 mesenchymal stem cells act as in vitro models for the precursor cells recruited to a natural defect.
  • [0044]
    We employed the same assay to test the bioactivity of BP released from 1% loaded nanospheres. BP was eluted from nanospheres at 37° C. in a 5% CO2 humidified incubator. After 24 hours 16% BP is released; and between 24 hours and 7 days, 7% BP was released (FIG. 2). The supernatant was collected, serial dilutions were made, and the supernatant was added to 10T1/2 micromass cultures. BP released from nanospheres at both time points formed spheroids at concentrations greater than 20 ng/ml, but not at concentrations between 0 and 10 ng/ml (see following table). Non-encapsulated BP also formed spheroids at concentrations greater than 20 ng/ml, but not at concentrations between 0 and 10 ng/ml. We conclude that both nanosphere encapsulation and release of BP does not inhibit BP bioactivity.
  • [0045]
    Spheroid formation (−=no spheroid formation; +=spheroid formation):
    BP concentration (ng/ml)
    state of used BP 0-10 20-1000
    non-encapsulated BP +
    released from nanospheres (24 h) +
    released from nanospheres (168 h) +
  • [0046]
    To determine the effect of BP slow release in the direct presence of micromass cultures, the following assay was performed. Nanospheres were washed for 24 hours and the supernatant was discarded. The nanospheres were then added to micromass cultures at a quantity such that 10 or 25 ng/ml of BP would be released over 24 hours. Release of 25 ng/ml resulted in spheroid formation whereas release of 10 ng/ml did not form spheroids (FIG. 5). Similarly, the addition of 10 ng of non-encapsulated BP per ml did not form a spheroid whereas the addition of 25 ng of non-encapsulated BP per ml did form a spheroid. Regarding the specific in vitro set-up, we conclude that slow release of BP over 24 hours is as effective as a single dose of BP.
  • [0047]
    To determine whether the BP released from nanospheres was as chondrogenic as non-encapsulated BP, spheroids were analyzed for type II collagen and proteoglycan content. 10T1/2 spheroids from the above assay that had formed with 1 μg of released BP per ml or 1 μg of non-encapsulated BP per ml were tested histologically with Azure and H+E stains and immunocytochemically with antibodies to type II collagen after 7 days. Both encapsulated and non-encapsulated BP induced cartilage markers such as type II collagen, proteoglycan, and round cell shape (FIG. 6). In addition, no qualitative differences were observed between encapsulated and non-encapsulated BP with respect to cell quantity, viability, morphology, or organization (FIG. 6). We conclude that BP retains full chondrogenic capacity after release from nanospheres.
  • [0048]
    The in vitro models used for determining the chondroinductive effect of BP differ from the in vivo case by the fact that in the in vitro case the precursor cells are present in an appropriate number and in an appropriate distribution whereas in the in vivo case the precursor cells first have to populate the defect and for this reason have to migrate into the defect. Only in the latter case and for achieving repair cartilage which resembles natural cartilage to a high degree, it is essential for the BP to be released over a prolonged time period according to a specific release profile.
  • EXAMPLE
  • [0049]
    The following example shows that BP released from nanospheres induces cartilage repair in chondral defects in vivo whereby virtually all cells recruited to the defect become chondrocytes, whereby the cell structure obtained is ordered, and whereby a hyaline matrix is built up.
  • [0050]
    Using a sheep model, unilateral defects of 0.5 mm width, 0.5 mm depth and 8 to 10 mm length were created in the trochlear groove of the patella. The defects did not penetrate the subchondral bone. The sheep employed in this study were seven years old and displayed degenerative symptoms, including brittle bones, chondromalacia, and subchondral cysts. Because of their advanced age and degenerative symptoms, these amimals probably have decreased numbers of precursor cells. The defects were then dressed according to Hunziker and Rosenberg (J. Bone Joint Surg. 78A(5): 721-733, 1996) with minor changes. Briefly, after enzymatic proteoglycan removal with Chondroitinase AC, 2.5 μl of a solution containig 200 units Thrombin per ml was placed in the defect. Then, a paste was filled into the defect, the paste containing per ml: 60 mg Sheep Fibrinogen (Sigma), 88 mg Gelfoam (Upjohn) and either 10 μg of BP-nanospheres or 10 μg of BP-nanospheres plus 80 ng rhIGF-1 (R+D Systems).
  • [0051]
    The nanospheres used were the nanospheres as specified in the description regarding FIG. 2 and they were loaded with 1% (w/w) of BP.
  • [0052]
    Assuming that the in vitro determined release rate is approximately the same as for the in vivo case, 10 to 20 ng BP per ml were released during the first 24 hours and approximately 0.1 to 1 ng per day for the following approximately 60 days.
  • [0053]
    After eight weeks, necropsies were performed. The repaired cartilage histology showed that virtually all of the precursor cells were differentiated to chondrocytes throughout the defect. In addition, there was an ordered cartilage appearance with cells on the top being more flattened morphologically than cells in the center and with the presence of ordered, stacked chondrocytes in the lowest zone. The repaired cartilage was fully integrated into the endogenous tissue. In addition, the cartilage repaired with only BP-nanospheres was not significantly different from the cartilage repaired using BP-nanospheres plus IGF-1.
  • [0054]
    In conclusion, these results demonstrate that BP released from nanospheres is sufficient for cartilage repair and that no addintional factor is required (such as e.g recombinant factor IGF-1). Using the inventive device constitutes a one step method for cartilage repair, whereby the nanosphere release of BP is sufficient for differentiation of virtually all of the precursor cells to chondrocytes and for induction of an ordered cartilage structure.
  • [0000]
    Other Publications:
  • [0055]
    Archer C W, Morrison E H, Bayliss M T, Ferguson M W: The development of articular cartilage: II. The spatial and temporal patterns of glycosaminoglycans and small leucine-rich proteoglycans; J Anat (ENGLAND) 189 (Pt 1): 23-35 (1996)
  • [0056]
    Atkinson B L, Fantle, K S, Benedict J J, Huffer W E, Gutierrez-Hartmann A: A Combination of Osteoinductive Bone Proteins Differentiates Mesenchymal C3H/10T1/2 Cells Specifically to the Cartilage Lineage; J. Cellular Biochem. 65: 325-339 (1997).
  • [0057]
    Celeste A J, Iannazzi J A, Taylor R C, Hewick R M, Rosen V, Wang E A, Wozney J M: Identification of transforming growth factor beta family members present in bone-inductive protein purified from bovine bone; Proc Natl Acad Sci U S A, Dec, 87(24): 9843-7 (1990)
  • [0058]
    Denker A E, Nicoll S B, Tuan R S: Formation of cartilage-like spheroids by micromass cultures of murine C3H10T1/2 cellls upon treatment with transforming growth factor β1′; Differentiation 59(1): 25-34 (1995)
  • [0059]
    Denker A E, Nicoll S B, Tuan R S: 41st Annual Meeting Orthop. Res. Society. (abstract): 465 (1995)
  • [0060]
    Flechtenmacher J, Huch K, Thonar E J, Mollenhauer J A, Davies S R, Schmid T M, Puhl W, Sampath T K, Aydelotte M B, Kuettner K E: Recombinant human osteogenic protein 1 is a potent stimulator of the synthesis of cartilage proteoglycans and collagens by human articular chondrocytes; Arthritis Rheum, Nov, 39(11): 1896-904 (1996)
  • [0061]
    Hunziker E B and Rosenberg L C: Repair of Partial-Thickness Defects in Articular Cartilage: Cell Recruitment from the Synovial Membrane; J. Bone Joint Surgery 78-A(5): 721-733 (1996)
  • [0062]
    Kim S, Turker M S, Chi E Y, Sela S, Martin G M: Preparation of multivesicular liposomes; Bioch. et Biophys. Acta 728:339-348 (1983)
  • [0063]
    Landry F B: Degradation of Poly (D,L-lactic acid) Nanoparticles in artificial gastric and intestinal fluids; in vivo uptake of the nanoparticles and their degradation products; Thesis for the Dept. of Biochemistry, Pharmacy, and Food Chemistry of the Johann Wolfgang Goethe University in Frankfurt, Germany
  • [0064]
    Luyten F P, Cunningham N S, Ma S, Muthukumaran N, Hammonds R G, Nevins W B, Woods W I, Reddi A H: Purification and partial amino acid sequence of osteogenin, a protein initiating bone differentiation; J Biol Chem, 264(23): 13377-80 (1989)
  • [0065]
    Massague J: TGFβ Signaling: Receptors, Transducer, and Mad Proteins; Cell 85: 947-950 (1996)
  • [0066]
    Mathiowitz E, Jacob J S, Jong Y S, Carino G P, Chickering D E, Chaturvedi P, Santos C A, Vijayaraghavan K, Montgomery S, Bassett M, Morrell C: Biologically erodable microspheres as potential oral drug delivery systems; Nature 386: 410-4 (1997)
  • [0067]
    Morrison E H, Ferguson M W, Bayliss M T, Archer C W: The development of articular cartilage: I. The spatial and temporal patterns of collagen types; J Anat (ENGLAND) 189(Pt 1): 9-22 (1996)
  • [0068]
    Mow V C, Ratcliff A, Poole A R: Cartilage and diarthrodial joints as paradigms for hierarchical materials and stuctures; Biomaterials 13(2): 67-97 (1992) Sailor L Z, Hewick R M, Morris E A: Recombinant human bone morphogenetic Protein-2 maintains the articular chondrocyte phenotype in long-term culture; J. Orthop. Res. 14: 937-945 (1996)
  • [0069]
    Urist MR: Bone: formation by autoinduction; Science 150: 893-899 (1965)
  • [0070]
    Wang E A, Rosen V, D'Alessandro J S, Bauduy M, Cordes P, Harada T, Israel D I, Hewick R M, Kerns K M, LaPan P, Luxenberg D P, McQuaid D, Moutsatsos I, Nove J, Wozney J M: Recombinant human bone morphogenetic protein induces bone formation; Proc Natl Acad Sci U S A, 87(6): 2220-4 (1990)
  • [0071]
    Wozney J M, Rosen V, Celeste A J, Mitsock L M, Whitters M J, Kriz R W, Hewick R M, Wang E A: Novel Regulators of bone formation: molecular clones and activities; Science 242: 1528-34 (1988)
Patentzitate
Zitiertes PatentEingetragen Veröffentlichungsdatum Antragsteller Titel
US3318774 *22. Juni 19659. Mai 1967Squibb & Sons IncTreatment of osseous and other tissue
US3458397 *8. Dez. 196629. Juli 1969Squibb & Sons IncProcess for producing osteogenic material
US3867728 *5. Apr. 197325. Febr. 1975Cutter LabProsthesis for spinal repair
US4002602 *22. Aug. 197511. Jan. 1977Gideon GoldsteinUbiquitous immunopoietic polypeptide (UBIP) and methods
US4394370 *21. Sept. 198119. Juli 1983Jefferies Steven RBone graft material for osseous defects and method of making same
US4434094 *12. Apr. 198328. Febr. 1984Collagen CorporationPartially purified osteogenic factor and process for preparing same from demineralized bone
US4440750 *12. Febr. 19823. Apr. 1984Collagen CorporationOsteogenic composition and method
US4455256 *5. Mai 198119. Juni 1984The Regents Of The University Of CaliforniaBone morphogenetic protein
US4529290 *16. Aug. 198416. Juli 1985Minolta Camera Kabushiki KaishaOperation mode display device for picture taking devices
US4563350 *24. Okt. 19847. Jan. 1986Collagen CorporationInductive collagen based bone repair preparations
US4596574 *14. Mai 198424. Juni 1986The Regents Of The University Of CaliforniaBiodegradable porous ceramic delivery system for bone morphogenetic protein
US4608199 *20. März 198426. Aug. 1986Arnold CaplanBone protein purification process
US4637931 *9. Okt. 198420. Jan. 1987The United States Of America As Represented By The Secretary Of The ArmyPolyactic-polyglycolic acid copolymer combined with decalcified freeze-dried bone for use as a bone repair material
US4663358 *25. Apr. 19865. Mai 1987Biomaterials Universe, Inc.Porous and transparent poly(vinyl alcohol) gel and method of manufacturing the same
US4678470 *29. Mai 19857. Juli 1987American Hospital Supply CorporationBone-grafting material
US4681763 *11. Juni 198521. Juli 1987University Of Medicine And Dentistry Of New JerseyComposition for stimulating bone growth
US4743259 *29. Okt. 198610. Mai 1988The University Of Virginia Alumni Patents FoundationUse of demineralized bone matrix in the repair of segmental defects
US4761471 *22. Aug. 19862. Aug. 1988The Regents Of The University Of CaliforniaBone morphogenetic protein composition
US4795467 *4. Apr. 19863. Jan. 1989Collagen CorporationXenogeneic collagen/mineral preparations in bone repair
US4795804 *7. Aug. 19853. Jan. 1989The Regents Of The University Of CaliforniaBone morphogenetic agents
US4801299 *22. Febr. 198431. Jan. 1989University Patents, Inc.Body implants of extracellular matrix and means and methods of making and using such implants
US4804744 *5. Sept. 198614. Febr. 1989International Genetic Engineering, Inc.Osteogenic factors
US4810691 *10. Dez. 19877. März 1989Collagen CorporationPolypeptide cartilage-inducing factors found in bone
US4834757 *28. März 198830. Mai 1989Brantigan John WProsthetic implant
US4843063 *8. Juni 198827. Juni 1989Collagen CorporationPolypeptide cartilage-inducing factors found in bone
US4902296 *29. Okt. 198720. Febr. 1990The University Of Virginia Alumni Patents FoundationUse of demineralized bone matrix in the repair of segmental defects
US4904260 *25. Juli 198827. Febr. 1990Cedar Surgical, Inc.Prosthetic disc containing therapeutic material
US4950483 *16. Dez. 198821. Aug. 1990Collagen CorporationCollagen wound healing matrices and process for their production
US4952404 *19. Juni 198728. Aug. 1990President And Fellows Of Harvard CollegePromotion of healing of meniscal tissue
US4992226 *16. Sept. 198812. Febr. 1991Collagen CorporationMethod of making molds with xenogeneic collagen/mineral preparations for bone repair
US5001169 *6. Jan. 198619. März 1991Collagen CorporationInductive collagen-based bone repair preparations
US5002583 *8. Aug. 198626. März 1991Sandu PitaruCollagen implants
US5015255 *10. Mai 198914. Mai 1991Spine-Tech, Inc.Spinal stabilization method
US5100422 *26. Mai 198931. März 1992Impra, Inc.Blood vessel patch
US5108438 *7. Mai 199028. Apr. 1992Regen CorporationProsthetic intervertebral disc
US5116738 *26. Apr. 199126. Mai 1992Genetics Institute, Inc.DNA sequences encoding
US5118667 *3. Mai 19912. Juni 1992Celtrix Pharmaceuticals, Inc.Bone growth factors and inhibitors of bone resorption for promoting bone formation
US5141905 *17. Nov. 198925. Aug. 1992Rosen Vicki ADna sequences encoding bmp-7 proteins
US5187076 *7. März 199016. Febr. 1993Genetics Institute, Inc.DNA sequences encoding BMP-6 proteins
US5192326 *9. Sept. 19919. März 1993Pfizer Hospital Products Group, Inc.Hydrogel bead intervertebral disc nucleus
US5206023 *31. Jan. 199127. Apr. 1993Robert F. ShawMethod and compositions for the treatment and repair of defects or lesions in cartilage
US5208219 *14. Febr. 19914. Mai 1993Celtrix Pharmaceuticals Inc.Method for inducing bone growth
US5219576 *3. Dez. 199115. Juni 1993Collagen CorporationCollagen wound healing matrices and process for their production
US5290763 *22. Apr. 19911. März 1994Intermedics Orthopedics/Denver, Inc.Osteoinductive protein mixtures and purification processes
US5306311 *17. Dez. 199126. Apr. 1994Regen CorporationProsthetic articular cartilage
US5314477 *4. März 199124. Mai 1994J.B.S. Limited CompanyProsthesis for intervertebral discs and instruments for implanting it
US5322933 *7. Mai 199221. Juni 1994The United States Of America As Represented By The Secretary Of The Department Of Health And Human ServicesCrystal structure of TGF-β-2
US5387213 *20. Aug. 19937. Febr. 1995Safir S.A.R.L.Osseous surgical implant particularly for an intervertebral stabilizer
US5390683 *21. Febr. 199221. Febr. 1995Pisharodi; MadhavanSpinal implantation methods utilizing a middle expandable implant
US5393739 *15. Sept. 199328. Febr. 1995Celtrix Pharmaceuticals, Inc.Use of bone morphogenetic protein in synergistic combination with TGF-β for bone repair
US5425772 *20. Sept. 199320. Juni 1995Brantigan; John W.Prosthetic implant for intervertebral spinal fusion
US5510121 *4. Mai 199523. Apr. 1996Rhee; Woonza M.Glycosaminoglycan-synthetic polymer conjugates
US5514180 *14. Jan. 19947. Mai 1996Heggeness; Michael H.Prosthetic intervertebral devices
US5543392 *25. Febr. 19936. Aug. 1996Morinaga Milk Industry Co., Ltd.Digestive tract cell activating agent of EGF and lactoferrin
US5591234 *19. Sept. 19957. Jan. 1997Axel KirschPost-surgery orthopedic covering
US5595722 *7. Juni 199521. Jan. 1997Neorx CorporationMethod for identifying an agent which increases TGF-beta levels
US5616490 *4. Mai 19951. Apr. 1997Ribozyme Pharmaceuticals, Inc.Ribozymes targeted to TNF-α RNA
US5629009 *7. Aug. 199613. Mai 1997Massachusetts Institute Of TechnologyDelivery system for controlled release of bioactive factors
US5632747 *15. März 199527. Mai 1997Osteotech, Inc.Bone dowel cutter
US5645597 *29. Dez. 19958. Juli 1997Krapiva; Pavel I.Disc replacement method and apparatus
US5705477 *5. Juli 19946. Jan. 1998The United States Of America As Represented By The Department Of Health And Human ServicesCompositions of transforming growth factor β(TGF-β) which promotes wound healing and methods for their use
US5707962 *28. Sept. 199413. Jan. 1998Gensci Regeneration Sciences Inc.Compositions with enhanced osteogenic potential, method for making the same and therapeutic uses thereof
US5716416 *10. Sept. 199610. Febr. 1998Lin; Chih-IArtificial intervertebral disk and method for implanting the same
US5718707 *22. Jan. 199717. Febr. 1998Mikhail; W. E. MichaelMethod and apparatus for positioning and compacting bone graft
US5723331 *6. Juni 19953. März 1998Genzyme CorporationMethods and compositions for the repair of articular cartilage defects in mammals
US5741685 *7. Juni 199521. Apr. 1998Children's Medical Center CorporationParenchymal cells packaged in immunoprotective tissue for implantation
US5786217 *14. Apr. 199728. Juli 1998Genzyme CorporationMethods and compositions for the repair of articular cartilage defects in mammals
US5865846 *15. Mai 19972. Febr. 1999Bryan; VincentHuman spinal disc prosthesis
US5902785 *7. Mai 199611. Mai 1999Genetics Institute, Inc.Cartilage induction by bone morphogenetic proteins
US5904716 *26. Apr. 199518. Mai 1999Gendler; ElMethod for reconstituting cartilage tissue using demineralized bone and product thereof
US5908784 *15. Nov. 19961. Juni 1999Case Western Reserve UniversityIn vitro chondrogenic induction of human mesenchymal stem cells
US5919235 *30. Sept. 19966. Juli 1999Sulzer Orthopaedie AgIntervertebral prosthesis
US5928940 *8. Okt. 199627. Juli 1999Creative Biomolecules, Inc.Morphogen-responsive signal transducer and methods of use thereof
US6010698 *24. März 19984. Jan. 2000Campina Melkunie B. V.Process for recovering growth factors, or a composition containing one or more growth factors, from milk or a milk derivative
US6013853 *15. Febr. 199411. Jan. 2000The University Of Texas SystemContinuous release polymeric implant carrier
US6042610 *24. Febr. 199828. März 2000Regen Biologics, Inc.Meniscal augmentation device
US6054122 *7. Juni 199525. Apr. 2000The American National Red CrossSupplemented and unsupplemented tissue sealants, methods of their production and use
US6080579 *26. Nov. 199727. Juni 2000Charlotte-Mecklenburg Hospital AuthorityMethod for producing human intervertebral disc cells
US6177406 *5. Sept. 199723. Jan. 2001Genetics Institute, Inc.BMP-3 products
US6179871 *14. Nov. 199630. Jan. 2001Alan A. HalpernMeans for cartilage repair
US6206923 *8. Jan. 199927. März 2001Sdgi Holdings, Inc.Flexible implant using partially demineralized bone
US6211157 *16. Okt. 19983. Apr. 2001Sulzer Biologics, Inc.Protein mixtures to induce therapeutic angiogenesis
US6242247 *2. Juni 19975. Juni 2001Sulzer Orthopedics Ltd.Method for making cartilage and implants
US6245107 *28. Mai 199912. Juni 2001Bret A. FerreeMethods and apparatus for treating disc herniation
US6251143 *4. Juni 199926. Juni 2001Depuy Orthopaedics, Inc.Cartilage repair unit
US6340369 *14. Aug. 200022. Jan. 2002Bret A. FerreeTreating degenerative disc disease with harvested disc cells and analogues of the extracellular matrix
US6344058 *14. Aug. 20005. Febr. 2002Bret A. FerreeTreating degenerative disc disease through transplantation of allograft disc and vertebral endplates
US6352557 *14. Aug. 20005. März 2002Bret A. FerreeTreating degenerative disc disease through transplantion of extracellular nucleus pulposus matrix and autograft nucleus pulposus cells
US6372257 *29. Juni 200016. Apr. 2002J. Alexander MarchoskyCompositions and methods for forming and strengthening bone
US6413511 *6. Juni 19952. Juli 2002University Of Pittsburgh Of The Commonwealth System Of Higher EducationCartilage alterations by administering to joints chondrocytes comprising a heterologous polynucleotide
US6419702 *14. Aug. 200016. Juli 2002Bret A. FerreeTreating degenerative disc disease through transplantation of the nucleus pulposis
US6511958 *16. Febr. 200028. Jan. 2003Sulzer Biologics, Inc.Compositions for regeneration and repair of cartilage lesions
US6514514 *16. Febr. 19994. Febr. 2003Sùlzer Biologics Inc.Device and method for regeneration and repair of cartilage lesions
US6558949 *13. Dez. 20006. Mai 2003Byoung-Hyun MinMedia for culturing human cells comprising human serum and method for culturing human cells using the same
US6582471 *12. Aug. 199824. Juni 2003Sulzer Innotec AgComposition and device for in vivo cartilage repair
US6723335 *7. Apr. 200020. Apr. 2004Jeffrey William MoehlenbruckMethods and compositions for treating intervertebral disc degeneration
US20020009789 *19. März 200124. Jan. 2002Jcr Pharmaceuticals Co., Ltd.Powder containing physiologically active peptide
US20020040004 *22. Dez. 20004. Apr. 2002Benedict James J.Method of promoting natural bypass
US20030135209 *4. Dez. 200017. Juli 2003Bahaa SeedhomFixation technology
Klassifizierungen
US-Klassifikation424/425, 623/23.61
Internationale KlassifikationA61F2/00, A61F2/30, A61F2/02, A61L27/54, A61P19/00, A61L27/00, A61K9/51, A61F2/28
UnternehmensklassifikationY10S977/91, Y10S977/914, A61F2/30756, A61F2210/0004, A61F2002/30036, A61K9/5153, A61L27/24, A61L27/26, A61L2300/624, A61L2430/06, A61B17/06166, A61F2002/2817, A61L2300/30, A61F2310/00365, A61L27/54, A61L27/50, A61L27/48, A61F2250/0035, A61L2300/252, B82Y5/00, A61F2002/30677, A61L27/227, A61L2400/12, A61F2002/30062
Europäische KlassifikationA61K9/51H6D4, A61F2/30C, A61L27/24, A61L27/22R, B82Y5/00, A61L27/26, A61L27/48, A61L27/50, A61L27/54