WO1999008728A1 - Composition and device for in vivo cartilage repair - Google Patents
Composition and device for in vivo cartilage repair Download PDFInfo
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- WO1999008728A1 WO1999008728A1 PCT/EP1998/005100 EP9805100W WO9908728A1 WO 1999008728 A1 WO1999008728 A1 WO 1999008728A1 EP 9805100 W EP9805100 W EP 9805100W WO 9908728 A1 WO9908728 A1 WO 9908728A1
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- nanospheres
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Definitions
- Articular cartilage an avascular tissue found at the ends of articulating bones, has no natural capacity to heal.
- 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., 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.
- BMPs Bone Morphogenetic Proteins
- BP Bactet Protein
- BMP and TGF ⁇ molecules bind to cell surface receptors (the BMP/TGF ⁇ 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).
- Urist described a substantially pure, but not recombinant BMP, combined with a biodegradable polylactic acid polymer delivery system for bone repair (US-4,563,489). This system blends together equal quantities of BMP and polylactic acid (PLA) powder (100 ⁇ g of each) and decreases the amount of BMP required to promote bone repair.
- BMP polylactic acid
- Hunziker (US-5,368,858; US-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.
- a specific concentration of proliferation/chemotactic agent fills the defect with repair cells.
- a larger transforming factor concentration transforms repair cells into chondrocytes.
- the proliferation agent and the transforming agent may both be TGF ⁇ differing in concentration only.
- the patent discloses a liposome encapsulation method for delivering TFG ⁇ -1 serving as transformation agent.
- 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.
- BMP-9 is encapsulated in a resorbable polymer system and delivered to coincide with the presence of cartilage formation inducing protein (s).
- 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.
- 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/TGF ⁇ 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.
- 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.
- 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 Figure 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.
- 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).
- 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.
- 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.
- factors e.g. derived from natural tissue
- 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).
- 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.
- nanospheres 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.
- 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/TGF ⁇ 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.
- the nanospheres 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 TGF ⁇ receptors to provide localized highly efficient delivery. The precursor cells become differentiated to chondrocytes and secrete type II collagen and cartilage-specific proteoglycans.
- 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.
- 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.
- inventive composition without matrix
- injection can be envisaged also, in particular in the case of small defects.
- at least 2 ⁇ g of the composition per ml of defect size is applied or at least 20ng of the osteoinductive and/or chondroinductive mixture encapsulated in the nanospheres is applied per ml defect size.
- 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.
- 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.
- Figure 1 shows a scanning electron micrograph of BP-loaded nanospheres
- Figure 2 shows the release profile (cumulative release vs. time) of the inventive composition
- Figure 3 shows the release profile of the inventive composition compared with release profiles of nanosphere delivery systems loaded with other proteins
- Figure 4 shows the volume of a cartilage defect vs. the days required for populating the defect with repair cells;
- Figure 5 shows micromass cultures in the presence or absence of nanospheres loaded with BP;
- Figure 6 shows cartilage marker analyses for in vitro cultures containing BP only and for similar cultures containing the inventive composition
- Figure 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.
- 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.
- 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).
- BCA assay Pieris-Coupled Reassay
- 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).
- the release is intermediate between zero-order and first-order kinetics. Both the 1% and 4% encapsulated BP have similar release profiles.
- the nanospheres are to be adapted accordingly when using factor mixtures other than BP.
- 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.
- 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.
- 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.
- 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).
- patients with larger quantities of progenitor cells and/ or smaller defects may require a shorter release rate profile.
- Figure 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.
- BSA bovine serum albumin
- PIBP bovine bone derived proteins
- Figure 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.
- the specific delivery system nanospheres as specified above encapsulating the factors
- BP specific protein mixture
- BP was eluted from nanospheres at 37°C in a 5% CO 2 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.
- 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.
- spheroids were analyzed for type II collagen and proteoglycan content. 10T1/2 spheroids from the above assay that had formed with 1 ⁇ g of released BP per ml or 1 ⁇ g 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, proteo- glycan, 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.
- 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.
- 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.
- the nanospheres used were the nanospheres as specified in the description regarding Figure 2 and they were loaded with 1% (w/w) of BP.
- Denker AE, Nicoll SB, Tuan RS Formation of cartilage-like spheroids by micromass cultures of murine C3H10T1/2 cellls upon treatment with transforming growth factor ⁇ l'; Differentiation 59(1): 25-34 (1995) Denker AE, Nicoll SB, Tuan RS: 41st Annual Meeting Orthop. Res. Society. (abstract): 465 (1995)
- Landry FB 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
- Luyten FP Cunningham NS, Ma S, Muthukumaran N, Hammonds RG, Nevins WB, Woods WI, Reddi AH: Purification and partial amino acid sequence of osteogenin, a protein initiating bone differentiation; J Biol Chem, 264(23): 13377-80 (1989)
- Urist MR Bone: formation by autoinduction; Science 150: 893-899 (1965) Wang EA, Rosen N, D'Alessandro JS, Bauduy M, Cordes P, Harada T, Israel DI, Hewick RM, Kerns KM, LaPan P, Luxenberg DP, McQuaid D, Moutsatsos I, ⁇ ove J, Wozney JM: Recombinant human bone morphogenetic protein induces bone formation;' Proc ⁇ atl Acad Sci U S A, 87(6): 2220-4 (1990) Wozney JM, Rosen N, Celeste AJ, Mitsock LM, Whitters MJ, Kriz RW, Hewick RM, Wang EA: Novel Regulators of bone formation: molecular clones and activities; Science 242: 1528-34 (1988)
Abstract
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Priority Applications (10)
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JP2000509464A JP2001514935A (en) | 1997-08-14 | 1998-08-12 | Compositions and devices for in vivo cartilage repair |
US09/485,594 US6582471B1 (en) | 1997-08-14 | 1998-08-12 | Composition and device for in vivo cartilage repair |
CA002300415A CA2300415C (en) | 1997-08-14 | 1998-08-12 | Composition and device for in vivo cartilage repair |
AU92607/98A AU746151B2 (en) | 1997-08-14 | 1998-08-12 | Composition and device for in vivo cartilage repair |
US09/250,370 US6514514B1 (en) | 1997-08-14 | 1999-02-16 | Device and method for regeneration and repair of cartilage lesions |
US09/505,209 US6511958B1 (en) | 1997-08-14 | 2000-02-16 | Compositions for regeneration and repair of cartilage lesions |
US10/386,946 US20030236574A1 (en) | 1997-08-14 | 2003-03-11 | Composition and device for in vivo cartilagerepair |
US11/046,674 USRE41286E1 (en) | 1997-08-14 | 2005-01-28 | Compositions for regeneration and repair of cartilage lesions |
US11/433,968 US20060216325A1 (en) | 1997-08-14 | 2006-05-15 | Composition and device for in vivo cartilagerepair |
US12/463,108 US20090297605A1 (en) | 1997-08-14 | 2009-05-08 | Composition And Device For In Vivo Cartilage Repair |
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EP97810567A EP0896825B1 (en) | 1997-08-14 | 1997-08-14 | Composition and device for in vivo cartilage repair comprising nanocapsules with osteoinductive and/or chondroinductive factors |
EP97810567.4 | 1997-08-14 |
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US10/386,946 Continuation US20030236574A1 (en) | 1997-08-14 | 2003-03-11 | Composition and device for in vivo cartilagerepair |
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US20030236574A1 (en) | 2003-12-25 |
AU9260798A (en) | 1999-03-08 |
AU746151B2 (en) | 2002-04-18 |
US6582471B1 (en) | 2003-06-24 |
US20060216325A1 (en) | 2006-09-28 |
JP2001514935A (en) | 2001-09-18 |
EP0896825B1 (en) | 2002-07-17 |
DE69714035T2 (en) | 2003-03-06 |
ATE220564T1 (en) | 2002-08-15 |
CA2300415C (en) | 2009-10-27 |
DE69714035D1 (en) | 2002-08-22 |
US6514514B1 (en) | 2003-02-04 |
US20090297605A1 (en) | 2009-12-03 |
EP0896825A1 (en) | 1999-02-17 |
USRE41286E1 (en) | 2010-04-27 |
CA2300415A1 (en) | 1999-02-25 |
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