ANHYDROUS BIOCOMPATIBLE COMPOSITE MATERIALS

Abstract

The invention is directed to biocompatible composite materials for medical applications such as tissue regeneration. In particular, the present invention is directed to biocompatible composite materials that may be used for the treatment of lost bone or bone defects. According to the invention there is provided an anhydrous biocompatible composite material comprising a biodegradable polymeric material and a granular synthetic material, wherein the polymeric material essentially consists of at least one block copolymer that comprises at least one hydrophilic block and at least one hydrophobic block.

Claims

1. An anhydrous biocompatible composite material comprising a biodegradable polymeric material and a granular synthetic material, wherein the polymeric material consists essentially of at least one block copolymer, wherein said at least one block copolymer is a polymer of formula (I)
X.sub.n-B.sub.q-A.sub.p-B.sub.q-X.sub.m-[B.sub.q-A.sub.p-B.sub.q].sub.l (I) wherein; A and B are independently methylene oxide, ethylene oxide, propylene oxide, butylene oxide, dioxanone or phenyl oxide; X is a polyamide, polyester, polyurethane, polycarbonate or polyester unit; l is 0 or 1; m is 1 to 25; n is 0 to 25; p is 2 to 150; q is 0 to 100; and l+n is more than 0.

2. The anhydrous biocompatible composite material of claim 1, wherein l is 0 and n is m.

3. The anhydrous biocompatible composite material of claim 1, wherein l is 1 and n is 0.

4. The anhydrous biocompatible composite material of claim 1, wherein m is 2 to 10, and/or p is 6 to 100, and/or q is 0 to 50.

5. The anhydrous biocompatible composite material of claim 1, wherein the granular synthetic material is osteoconductive.

6. The anhydrous biocompatible composite material of claim 1, wherein the granular synthetic material comprises calcium phosphate.

7. The anhydrous biocompatible composite material of claim 1, wherein the actual ratio (n+m) to (p+q), as determined by .sup.1H NMR, is less than 0.36.

8. The anhydrous biocompatible composite material of claim 1, wherein m is n; l and q are 0.

9. The anhydrous biocompatible composite material of claim 1, that is an injectable, malleable and/or kneadable no-sticky putty that retains its shape at a typical temperature of 15 to 40 C.

10. The anhydrous biocompatible composite material of claim 9, that has been sterilized by -rays or electron beams.

11. A method to treat connective tissue and/or bone loss or defect which method comprises administering to a subject in need of such treatment the anhydrous biocompatible composite material of claim 1.

12. A method to sterilize a biodegradable polymeric material consisting essentially of one or more block copolymer comprising at least one hydrophilic block and at least one hydrophobic block, which method comprises irradiating the biodegradable polymeric material by -rays or electron beams.

13. A method to treat bone loss or defect, which method comprises shaping the anhydrous biocompatible composite material of claim 1 into a desired shape and placing said shaped material at the site of bone loss or defect.

14. The anhydrous biocompatible composite material of claim 1, wherein A and B are ethylene oxide or propylene oxide.

15. The anhydrous biocompatible composite material of claim 1, wherein X is a polyester unit.

16. The anhydrous biocompatible composite material of claim 1, wherein X is a hydroxybutyrate, lactic acid, glycolide, -butyrolactone, -valerolactone or -caprolactone.

17. The anhydrous biocompatible composite material of claim 16, wherein X is lactic acid.

18. The anhydrous biocompatible composite material of claim 4, wherein m is 3 to 7.

19. The anhydrous biocompatible composite material of claim 4, wherein p is 40 to 50.

20. The anhydrous biocompatible composite material of claim 4, wherein q is 0 to 19.

21. The anhydrous biocompatible composite material of claim 5, wherein the granular synthetic material is osteoinductive.

22. The anhydrous biocompatible composite material of claim 7, wherein the ratio (n+m) to (p+q), as determined by .sup.1H NMR, is less than 0.30.

23. The anhydrous biocompatible composite material of claim 22, wherein the ratio (n+m) to (p+q), as determined by .sup.1H NMR, is between 0.01 and 0.25.

24. The anhydrous biocompatible composite material of claim 23, wherein the ratio (n+m) to (p+q), as determined by .sup.1H NMR, is between 0.05 and 0.15.

25. The anhydrous biocompatible composite material of claim 7, wherein the ratio (n+m) to (p+q), as determined by .sup.1H NMR, is less than 0.10.

26. A method to engineer tissue to correct a connective tissue and/or bone defect which method comprises providing anhydrous biocompatible composite material of claim 1 to the defect.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0061] FIG. 1 shows results of dissolution the test for non-sterile samples in PBS.

[0062] FIG. 2 shows more results of the dissolution test for non-sterile samples in PBS. Legend labels of materials: (A) 25ML75HL; (B) 50ML50HL; (C) 50LL50ML; (E) 25LL75ML; (F) 25LL75HL; (G) 50LL50HL; (H) 50LH50HH; (I) 25LH75MH; (J) 50LH50MH

[0063] FIG. 3 shows one-week degradation of poly(propylene glycol)-based block copolymers (LPGL).

[0064] FIG. 4 shows that the PG4.sub.L-based putty quickly releases granules and forms a small polymer ball. As soon as the degrading medium was removed, the polymer ball lost shape. In contrast, PG4.sub.M- and PG4.sub.H-based putties kept the shape for the whole duration of the experiment (i.e. one week).

[0065] FIG. 5 illustrates the dissolution behavior of the selected putties, which seems similar to those not-sterile (FIG. 1).

[0066] FIG. 6A shows the initial shape and size of sterile putties. All have retained the granules after sterilization and kept the shape. However, as reported in table 15, they had different handling properties. FIG. 6B shows the putties after 32 minutes in PBS. Those putties with binders based on PEG started to disintegrate indicating a rapid triggering of binder dissolution. FIG. 6C shows the composite materials after one hour: all PEG-based putties released most of granules (observe the central block still standing: it will disappear during the next hour).

[0067] FIG. 7 shows a detailed microscope pictures for each composite material and histological analysis from dog 2. In (a) is the overview of putty A, (b) is overview of putty B, (c) is overview of putty F, and (d) is overview of putty MH. The scale bar in (a) applies for all overviews. Detailed images taken at higher magnification (x10) are shown in (e) for putty A, (f) for putty B, (g) for putty F and (h) for putty MH. Circles in (e), (f), (g) and (h) in indicate ceramic debris dispersed in soft tissue matrix. The boxes in (e), (f), (g) and (h) indicate where images with further higher magnification (20) were taken: (i) is putty A, (m) is putty B, (n) is putty F and (o) is putty MH. Letter M stands for material (i.e. CaP ceramic granules), black stars indicate osteoid, black arrows show the seam of osteoblast cells and gray arrows show multinucleated giant cells phagocytizing on the surface of the granules.

[0068] FIG. 8 shows the main endpoints of the dissolution test of the putties. All putties dissolved in an acceptable manner. Blend C and Blend W had faster dissolution leading to quicker putty disruption as compared to Blend A (PEGs with higher molecular weight) and Blend Z (presence of Pluronic P85, which presents hydrophobic poly(propylene glycol) in its structure).

[0069] FIG. 9 shows DSC curves for the three formulations: the decrease of melt temperature with the increase of lactide content is clearly visible.

[0070] FIG. 10 shows the physical appearance of the formulations according to the lactide content.

[0071] FIG. 11 shows the relationship between the actual ratio L/EO (i.e. the ratio lactic acid monomers (hydrophobic block) over ethylene oxide monomers (hydrophilic block)), the melting point range (Tm), PEG length and hardness of the putty is depicted. This figure shows that the actual ratio influences the physical properties of the putty.

EXAMPLE 1

[0072] In this example, the synthesis and characterization of a particular polymeric material in accordance with the present invention will be described.

1.1. Synthesis of poly(L-lactide)-co-poly(ethylene oxide)-co-poly(L-lactide) Triblock Block Copolymers (i.e. LEOL)

[0073] The LEOL block copolymers were synthesized via ring-open polymerization of L-lactide monomer in presence of poly(ethylene oxide) as initiator and stannous octoate (SnOct.sub.2) as catalyst. All reactions were performed in argon saturated atmosphere. Poly(ethylene oxide) (EO) and L-lactide monomer were blended in a glass three-necked flask and gradually warmed to 130 C. under gentle stirring under an argon atmosphere. Afterwards, 4-5 drops (ca. 160-170 L) of the catalyst were added and the mixture was let reacting at the temperature of 130 C. and stirring rate of 50 rpm for 24 hours. Table 2 shows the synthesized block copolymers.

TABLE-US-00002 TABLE 2 Poly(ethylene glycol)-based block copolymers (LEOL)* block ratio's used during the synthesis. Theoretical Copolymer ID block composition Theoretical L/EO mol. ratio LL L.sub.2.5EO.sub.23L.sub.2.5 0.22 ML L.sub.5EO.sub.46L.sub.5 HL L.sub.10EO.sub.91L.sub.10 HM L.sub.15EO.sub.91L.sub.15 0.33 LH L.sub.5EO.sub.23L.sub.5 0.44 MH L.sub.10EO.sub.46L.sub.10 HH L.sub.20EO.sub.91L.sub.20 *the XY nomenclature is used. The letter X indicates the EO block length (e.g. L = EO.sub.23; M = EO.sub.46; H = EO.sub.91) and Y indicates the L-lactic acid/EO block mol. ratio (e.g. L = 0.22; M = 0.33; H = 0.44). Wherein the theoretical L/EO molar ratio is the theoretical ratio as described herein above for the lactic acid monomers over the ethylene oxide monomers.

1.2. Characterization of Poly(Ethylene Glycol)-Based Block Copolymers (LEOL)

1.2.1. Physical (Gross) Appearance

[0074] The block copolymers were analyzed by a stereomicroscope to detect any undesired inhomogeneity. Afterwards the block copolymers were characterized for their hardness first via pressing with steel tools and subsequent shaping by hands. Table 3 summarizes the observations.

TABLE-US-00003 TABLE 3 Physical observations (handling) on poly(ethylene glycol)-based block copolymers (LEOL). ID Observations LL White soft mass that melts as soon as kept in hands leaving a sticky layer of polymer. ML Transparent thick but malleable mass. Does not melt as fast as LL in hands: there is need to process it to get it spread on skin leaving a sticky layer of polymer. HL Hard and waxy. Resists better when worked in hands and gets softer leaving an oily effect. HM Very similar to HL. Additional lactide (compared to HL) is expected to enhance handling and increase hydrophobicity. LH Extremely soft gel, which immediately gets liquid once touched with hands. MH Harder than ML (i.e. need to scratch) but gets melted quicker when worked in hands. HH Very hard and waxy. Similar to HL.

[0075] Based on these observations, a trend seems to exist wherein a shorter EO block results in a the softer mass (e.g. LL vs. ML vs. HL).

1.2.2. Melting Point: Visual Estimation

[0076] A volume of roughly one mL of each block copolymer was put in glass vials immersed in cold water bath, which was warmed via a gentle temperature ramp. The temperature of the block copolymer was continuously monitored and the melting point range was recorded: the lowest temperature was the one at which the polymer visually started to change state while the highest temperature was taken when the polymer was completely melt. Table 4 shows the results.

TABLE-US-00004 TABLE 4 Melting range of poly(ethylene glycol)-based block copolymers (LEOL). Melting point Appearance after Copolymer ID range [ C.] melting LL 30-35 Clear liquid ML 45-50 Clear liquid HL 55-60 Clear liquid HM 40-60 Clear liquid LH <30 Clear liquid MH <40 Clear liquid HH <40 Clear liquid

[0077] The results show that LEOL block copolymers have increasing melting temperature ranges with the increase of EO block size, but it decreases when lactide block size increased. Therefore, a few conclusions may be drawn: [0078] 1) the shorter EO block, the lower the melting point (e.g. LL vs. ML vs. HL); [0079] 2) the longer L-lactide block, the lower melting point (e.g. LL vs. LH and ML vs. MH).

1.2.3. Composition: .SUP.1.H-NMR

[0080] Copolymers were dissolved in deuterated chloroform (i.e. CDCl.sub.13, at any concentration) and spectra (NMR-spectra 1, vide infra) were obtained via proton nuclear magnetic resonance (.sup.1H-NMR, 400 MHz) to determine the molecular composition of each copolymer. Peak integration was performed on raw spectra (i.e. without any processing) and the total area of EO block peak (A.sub.EO) and the total area of two L-lactide block peaks (X.sub.L) were recorded. The final block composition was then calculated basing on the molecular formula X.sub.nEO.sub.pX.sub.n where p{23,46,91}, and using p:A.sub.EO=2 n:X.sub.L.

[0081] Table 5 compares the composition calculated from the measurements with those theoretical and reports the actual L/EO molecular ratio which can be calculated from the composition by .sup.1H-NMR: full synthesis of copolymers with higher lactide amounts was more difficult, leading to lower L/EO molecular ratios than those theoretical.

NMR-spectra-1. .sup.1H-NMR spectra's for the poly(ethylene glycol)-based block copolymers (LEOL). (a) LL, (b) ML, (c) HL, (d) LH, (e) MH and (f) HH. Indicated by arrow is the band for EO block (.sub.EO=3.55-3.80 ppm), while stars indicate the L-lactide block chemical shifts (.sub.L1=5.10-5.30 ppm, .sub.L2=1.40-1.70 ppm).

TABLE-US-00005 TABLE 5 Composition of poly(ethylene glycol)-based block copolymers (LEOL). Copolymer ID Theoretical Composition Actual L/EO composition by .sup.1H-NMR mol. ratio LL L.sub.2.5EO.sub.23L.sub.2.5 L.sub.2.3EO.sub.23L.sub.2.3 0.20 ML L.sub.5EO.sub.46L.sub.5 L.sub.4.3EO.sub.46L.sub.4.3 0.19 HL L.sub.10EO.sub.91L.sub.10 L.sub.8.1EO.sub.91L.sub.8.1 0.18 HM L.sub.15EO.sub.91L.sub.15 n/a n/a LH L.sub.5EO.sub.23L.sub.5 L.sub.4.5EO.sub.23L.sub.4.5 0.39 MH L.sub.10EO.sub.46L.sub.10 L.sub.9.1EO.sub.46L.sub.9.1 0.39 HH L.sub.20EO.sub.91L.sub.20 L.sub.16.3EO.sub.91L.sub.16.3 0.36 Wherein the actual L/EO molar ratio is the actual ratio as determined by .sup.1H NMR described herein above for the lactic acid monomers over the ethylene oxide monomers.

[0082] 1.3. Preparation of Blends of Poly(Ethylene Glycol)-Based Block Copolymers (LEOL)

[0083] Each block copolymer was mixed with another one in the desired proportions via syringe. Table 6 shows the blends prepared, including the calculated L/EO mol. ratio based on those obtained from .sup.1H-NMR data (Table 5). Please note that in a blend one cannot strictly speak of a L/EO molecular ratio, but it can be used as an indicator for the L-lactic acid amount in the final blend.

TABLE-US-00006 TABLE 6 Prepared blends of poly(ethylene glycol)-based block copolymers (LEOL). Composition [% v/v] Observations Theoretical L/EO mol. ratio 25LL75ML Malleable 0.19 50LL50ML 0.19 25LL75HL 0.18 50LL50HL 0.18 25ML75HL 0.18 50ML50HL 0.18 50ML50HM Malleable n/a (theoretical: 0.25) 25LH75MH Hard 0.39 50LH50MH 0.39 25LH75HH 0.36 50LH50HH 0.36 25MH75HH 0.36 50MH50HH 0.37

[0084] Longer EO blocks seem to result in harder to another one with shorter EO block, the resulting binder seems to be harder blends. Moreover, blends containing block copolymers with higher L-lactic acid/EO block molecular ratio are very hard. The melting point of resulting blends is not very different than that of the component with longer EO block.

EXAMPLE 2

Sterilization of Block Copolymers and Blends Thereof

[0085] The block copolymers and blends from Example 1 and listed below were sterilized with two different methods: -irradiation and e-beam. [0086] Copolymers: LL, ML, HL [0087] Blends: 25LL75HL, 25ML75HL, 50ML50HL

[0088] The materials were compared against their non-sterile counterparts on the basis of gross appearance (Table 7).

TABLE-US-00007 TABLE 7 Gross observations of block copolymers and blends after sterilization. ID Observations as compared to the non-sterile counterpart LL -irradiation: soft but thick cream (no observable changes). e-beam: soft but thick cream (no observable changes). ML -irradiation: hard mass (no observable changes). e-beam: hard mass (no observable changes). HL -irradiation: waxy mass (no observable changes). e-beam: waxy mass (no observable changes). 25LL75HL -irradiation: hard uniform mass (no observable changes). e-beam: hard uniform mass (no observable changes). 25ML75HL -irradiation: hard uniform mass (no observable changes). e-beam: hard uniform mass (no observable changes). 50ML50HL -irradiation: hard uniform mass (no observable changes). e-beam: hard uniform mass (no observable changes).

EXAMPLE 3

In Vitro Bench Testing of Formulation of LEOL Copolymer Binders with Particles Into Putty

3.1. Preparation of Putties

[0089] Based on positive handling evaluation, a subset of the block copolymer blends from Example 1 was selected as potential putty binders. Various putties were prepared with CaP ceramic granules (size 0.5-1 mm) and one binder in a binder/CaP volume ratio of 0.8. Accordingly, the polymer component was melt and mixed with CaP granules. Only those (non-sterile) putties that could retain granules have been further considered.

3.2. Characterization of Non-Sterile Putties

3.2.1. Blind Test

[0090] The blind test was executed by third persons to evaluate the handling performance of the putties, which were scored according to the guidelines as described herein above and in Table 1. The final score (FS) of each material was calculated, where the worst performance was scored with 5 and the best one with 30. The results are summarized in Table 8.

3.2.2. Dissolution Test

[0091] Dissolution test was done by immersing roughly 1 mL of each putty in 11 mL of phosphate buffered solution (i.e. PBS) at 37 C. without shaking. At the time points of 1, 2, 4, 8, 16 and >16 minutes pictures were taken. FIG. 1 and FIG. 2 show images of the dissolution during time, while Table 8 shows the results.

[0092] FIG. 1 shows results of dissolution the test for non-sterile samples in PBS.

[0093] All non-sterile LEOL-binders had a rapid start of dissolution (viz. within 30-60 minutes) and let the corresponding putties release granules. However, they had different dissolution rates as bulky bodies in the putties containing 25ML75HL, 50ML50HL, 25LL75HL, 50LL50HL, 50LH50HH and 25LH75MH were present after 32 min (see FIG. 2).

[0094] FIG. 2 shows more results of the dissolution test for non-sterile samples in PBS. Legend labels of materials: (A) 25ML75HL; (B) 50ML50HL; [0095] (C) 50LL50ML; (E) 25LL75ML; (F) 25LL75HL; (G) 50LL50HL; (H) 50LH50HH; (I) 25LH75MH; (J) 50LH50MH.

TABLE-US-00008 TABLE 8 Selected putties and results from the blind and dissolution tests. Composition binder/CaP vol ratio = 0.8 Blind test Dissolution test [min] l00LL n/a starts at 8 min, almost completely dissolved CaP granules at >16 min 100HL n/a starts at 16 min, almost completely dissolved CaP granules at >32 min 25LL75ML 21.4 starts at 16 min, partially dissolved at CaP granules >32 min 50LL50ML 20 starts at 1 min, partially dissolved at >16 min CaP granules 25LL75HL 24 starts at 16 min, almost completely dissolved CaP granules at >16 min 50LL50HL 22 starts at 16 min, almost completely dissolved CaP granules at >16 min 25ML75HL 23.6 partially dissolved at >16 min CaP granules 50ML50HL 20.8 partially dissolved at >16 min CaP granules 25LH75MH n/a (loss of starts at 2 min, almost completely dissolved CaP granules granules) at >16 min 50LH50MH n/a (loss of starts at 2 min, almost completely dissolved CaP granules granules) at >16 min 50LH50HH 22.2 the least dissolved at >16 min CaP granules

EXAMPLE 4

Copolymer Synthesis with Central Block Different Than PEG

[0096] Two families of L-lactide containing copolymers were prepared, differing in the type of their central block: one with poly(propylene glycol) (i.e. LPGL) and the other with Pluronic (LPUL). The central block could be with different molecular weight or different kinds of Pluronic. When the same central block was considered, different lactic acid/central block molar ratios were also considered.

4.1. Synthesis

[0097] All block copolymers were synthesized via ring-open polymerization of L-lactide monomer in presence of poly(propylene oxide) or Pluronic P65 as initiators and stannous octoate (i.e. SnOct.sub.2) as catalyst. All reactions were performed under an argon saturated atmosphere at 130 C. and stirring rate of 50 rpm for 24 hours. Table 9 shows the synthesized block copolymers.

TABLE-US-00009 TABLE 9 LPGL and PLUL block copolymers. Copolymer Design Theoretical L/PG Family ID Property mol. ratio LPGL PG4.sub.L L.sub.7.5PG.sub.68L.sub.7.5 0.22 poly(propylene glycol) PG4.sub.M L.sub.15PG.sub.68L.sub.15 0.44 based PG4.sub.H L.sub.22.5PG.sub.68L.sub.22.5 0.66 Copolymer Design Theoretical L/EO ID Property mol. ratio LPUL P65.sub.L L.sub.11P65L.sub.11 0.57 Pluronic based P65.sub.M L.sub.16.5P65L.sub.16.5 0.86 P65.sub.H L.sub.22P65L.sub.22 1.15 F127.sub.LL L.sub.11F127L.sub.11 0.11 F127.sub.M L.sub.87F127L.sub.87 0.87 Wherein the theoretical L/PG molar ratio is the theoretical ratio as described herein above for the lactic acid monomers over the ethylene oxide and propylene oxide monomers.

4.2. Characterization of Copolymers

4.2.1. Physical (Gross) Appearance of LPGL and LPUL Copolymers

[0098] The copolymers were observed with stereomicroscope to detect any presence of any undesired inhomogeneity. Afterwards they have been characterized regarding their hardness via pressing with steel tools first and with hands then. Table 10 summarizes the observations on LPGL copolymers and Table 11 refers to LPUL copolymers.

TABLE-US-00010 TABLE 10 Physical observations (handling) on poly(propylene glycole)-based block copolymers (LPGL). ID Notes PG4.sub.L At room temperature, viscous fluid tending to white color. PG4.sub.M Viscous and white semi-solid that quickly solidifies as soon as temperature drops from 130 C. PG4.sub.H White and thick fluid that quickly solidifies as soon as temperature drops from 130 C.

[0099] Basing on the gross observations, it may be said that the material hardens with the increase in L-lactide block size (e.g. PG4.sub.L vs. PG4.sub.M vs. PG4.sub.H).

TABLE-US-00011 TABLE 11 Physical observations (handling) on Pluronic-based block copolymers (LPUL). ID Notes P65.sub.L At room temperature is liquid. P65.sub.M At room temperature is liquid. P65.sub.H Soft mass F127.sub.LL Hard brownish solid mass. F127.sub.M Hard brownish solid mass.

4.2.2. Dissolution Test of LPGL Copolymers

[0100] Carefully weighed masses (m.sub.0) of PG4.sub.M and PG4.sub.H block copolymers were placed in 100 mL of distilled water, for one week at 371 C. and gently shaken. Three time points were considered (i.e. 1, 4 and 7 days; 1 replicate per time point), when the samples were harvested, excess water wiped away and their wet weight (mw) was measured. Then, each sample was photographed and then vacuum dried at 371 C. until stabilization of their weights. Therefore the dry weights (md) were taken. Fluid uptake (FU) and mass loss (ML) changes (in %) could be calculated, for each sample at each time point, as

FU=100*(m.sub.wm.sub.d)/m.sub.d

ML=100*(m.sub.0m.sub.d)/m.sub.0

[0101] The dimensional change is determined by the variation of average diameter (in %) during time, where the average diameter is given by the average of diameters measured along different directions. Table 12 show the results for fluid uptake, mass loss and average dimension changes (FIG. 3).

TABLE-US-00012 TABLE 12 One-week degradation of poly(propylene glycol)-based block copolymers (LPGL). Mass Loss Fluid Uptake Dimension [%] [%] Change [%] l 4 7 l 4 7 l 4 7 Copolymer day days days day days days day days days PG4M 1.6 1.5 2.6 4.9 3.5 3.0 17.1 7.5 2.6 PG4H 7.6 8.5 8.4 6.6 9.0 9.7 10.3 17.2 11.1

[0102] LPGL copolymers seem to change during time when in contact with PBS but they retain their bulky mass.

4.3. Preparation of LPGL- and LPUL-Based Putties and Dissolution Properties

[0103] Putties were prepared with CaP ceramic granules (size 0.5-1 mm) and one binder in a binder/CaP volume ratio of 0.8. Each putty underwent a dissolution test (see Example 3.2.2 for procedure): Table 13 and FIG. 4 show the results regarding LPGL-based putties.

TABLE-US-00013 TABLE 13 Physical observations (handling) on poly(propylene glycol)-based copolymer (LPGL) putties. ID Handling property Dissolution putty PG4L Loses granules, Granules were released, but polymer is not malleable. not dissolved. putty PG4M Very good Putty retains shape and size over a week handling. putty PG4H Good handling Putty retains shape and size over a week characteristics.

[0104] FIG. 4 shows that the PG4.sub.L-based putty quickly releases granules and forms a small polymer ball. As soon as the degrading medium was removed, the polymer ball lost shape. In contrast, PG4.sub.M- and PG4.sub.H-based putties kept the shape for the whole duration of the experiment (i.e. one week).

[0105] All LPUL-based putties were dissolvable in water and could release the ceramic granules (Table 14). It may be seen that those binders having less polylactide had a higher dissolution rate.

TABLE-US-00014 TABLE 14 Handling and dissolution properties of Pluronic-based block copolymer (LPUL) putties. ID Handling property Dissolution putty P65.sub.L Loses granules, Dissolved in 5 minutes, with granules not malleable. release. putty P65.sub.M Good handling. Dissolved in 1.5-2 hours, with granule release. putty P65.sub.H Brittle putty. Partially dissolved over 4 days.

EXAMPLE 5

Sterilization Putties

5.1 Sterilization of LEOL Putties

[0106] Certain LEOL putties from Example 3.1 were sterilized with -irradiation (25-40 kGy) and evaluated on their capacity to retain granules and be shaped. A subgroup was then selected basing on the handling results and subjected to dissolution test. FIG. 5 illustrates the dissolution behavior of the selected putties, which seems similar to those not-sterile (FIG. 1).

[0107] From the figures at time T=0 min, one can see which putties were still able to retain granules and be shaped after sterilization (i.e. putties with LL, 75LL25HL, 50LL50HL, 25LL75HL and HL).

[0108] Certain other putties from Example 3.1 were sterilized with -irradiation and compared against their non-sterile counterparts on the basis of gross shapeabling properties (Table 15), while their dissolution rate in PBS at 37 C. was evaluated as well (FIG. 6).

[0109] Certain putties (e.g. putty ML) showed even slightly improved handling characteristics after sterilization.

[0110] FIG. 6A shows the initial shape and size of sterile putties. All have retained the granules after sterilization and kept the shape. However, as reported in table 15, they had different handling properties. FIG. 6B shows the putties after 32 minutes in PBS. Those putties with binders based on PEG started to disintegrate indicating a rapid triggering of binder dissolution. FIG. 6C shows the composite materials after one hour: all PEG-based putties released most of granules (observe the central block still standing: it will disappear during the next hour). Pluronic- and poly(propylene glycol)-based putties were not disintegrated at all and did not release any granule after one hour. They will still keep the same shape after two days.

TABLE-US-00015 TABLE 15 Handling of certain putties after -irradiation. ID of putties (CaP/binder v/v ratio = 1.2-1.3) Observations as compared to the binder formulation non-sterile counterpart putty A (25ML75HL) Non-sterile: consistent mass, looks hard but it is shapeable After -irradiation: softer, with good shapeability and not sticky putty ML (100ML) Non-sterile: shapeable, softer than putty A After y-irradiation: good shapeability (even if a bit hard at the beginning) and not sticky putty HM (100HM) Non-sterile: hard and there is need to work it with hands before getting it shapeable After -irradiation: too hard putty NEW (50ML50HM) Non-sterile: consistent mass, looks hard but it is shapeable After -irradiation: harder but still shapeable

5.2 Sterilization of LPGL-Based Putties

[0111] Two preferred LPGL-based putties from Example 4 were sterilized with -irradiation (25-40 kGy). Table 16 shows the observations.

TABLE-US-00016 TABLE 16 Handling of poly(propylene glycol)-based copolymer (LPGL) putties after sterilization. ID Handling property after sterilization putty PG4.sub.M Non-sterile: excellent shaping properties After -irradiation: good shapeability, but a bit sticky. Retained granules and kept the shape, same handling properties were observed. putty PG4.sub.H Retained granules and kept the shape, same handling properties were observed. putty P65.sub.M Non-sterile: soft mass and sticky After -irradiation: too soft, with loss of granules

EXAMPLE 6

In Vivo Biological Performances of Selected LEOL Putties in Osteoinduction Model

[0112] Four LEOL putties from Example 3.1 (Table 17) were implanted in back muscle of five dogs for 8 weeks to evaluate the effect of four different LEOL binders on the osteoinduction potential of calcium phosphate ceramic granules (size 0.5-1 mm). After harvesting and histoprocessing, they have been stained with methylene blue/basic fuchsin and sectioned.

TABLE-US-00017 TABLE 17 The LEOL-based putties implanted. Material description Notes A putty 25ML75HL binder with CaP granules (0.5-1 mm) in polymer/CaP volume ratio of 0.8 B putty 50ML50HL binder with CaP granules (0.5-1 mm) in polymer/CaP volume ratio of 0.8 F putty 25LL75HL binder with CaP granules (0.5-1 mm) in polymer/CaP volume ratio of 0.8 MH putty 100MH binder with CaP granules (0.5-1 mm) in polymer/CaP volume ratio of 0.8

[0113] Not much difference was seen among the four composite materials, and histological observations are comparable with each other.

[0114] For all composite materials bone formation was observed, mainly scattered in spots over the whole implant area. Osteoid, with a seam of osteoblast cells lining its border surface, was present indicating active bone formation. CaP ceramic debris was occasionally observed as dispersed in soft tissue matrix in the form of small particles (dimensions in the order of tens of microns). Many multinucleated giant cells, with CaP ceramic nano-particles phagocytized, were present on the surface of ceramic granules. When no bone was observed (i.e. one B, one F and one MH), very limited amount of cells were seen and soft tissue looked like fibrous. No inflammatory signs were observed in any explant and no fibrotic capsule was formed.

[0115] Visually comparing the putty samples, not much difference was observed indicating that all four gels performed similarly. When the putties were compared to CaP ceramics alone, it appeared that bit less bone formation occurred indicating a possible slight bone-hindering effect of the gels. However, osteoid and osteoblast cells indicate that, after 8 weeks implantation, bone formation is still ongoing and may lead to a larger bone volume at later stages. The presence of multinucleated giant cells and of ceramic residuals dispersed in the soft tissue matrix, and the simultaneous absence of any inflammation signs, indicate cell-driven resorption of the ceramic granules is active.

[0116] FIG. 7 shows a detailed microscope pictures for each composite material and histological analysis from dog 2. In (a) is the overview of putty A, (b) is overview of putty B, (c) is overview of putty F, and (d) is overview of putty MH. The scale bar in (a) applies for all overviews. Detailed images taken at higher magnification (10) are shown in (e) for putty A, (f) for putty B, (g) for putty F and (h) for putty MH. Circles in (e), (f), (g) and (h) in indicate ceramic debris dispersed in soft tissue matrix. The boxes in (e), (f), (g) and (h) indicate where images with further higher magnification (20) were taken: (i) is putty A, (m) is putty B, (n) is putty F and (o) is putty MH. Letter M stands for material (i.e. CaP ceramic granules), black stars indicate osteoid, black arrows show the seam of osteoblast cells and gray arrows show multinucleated giant cells phagocytizing on the surface of the granules.

EXAMPLE 7

Selection of a Suitable Binder Formulation

[0117] In this example, additional blends of L-lactide/EO block copolymers were prepared and analyzed. The synthesis of the copolymers and the blends was carried out analogously to Example 1.1. The following blends of copolymers were prepared.

TABLE-US-00018 TABLE 18 Blends of copolymers Actual L/EO ID Formulation mol. ratio Blend A 26.4% wt. L-lactide + 18.4% wt. PEG2000 + 0.18 55.2% wt. PEG4000 Blend C 15% wt. L-lactide + 21.25% wt. PEG1000 + 0.10 63.75% wt. PEG2000 Blend W 15% wt. L-lactide + 23.4% wt. PEG1000 + 0.11 59.5% wt. PEG2000 + 2.1% wt. PEG3000 Blend Z 25% wt. L-lactide + 37.5% wt PLU85 + <0.05 37.5% wt PEG4000 Wherein the actual L/EO molar ratio is the actual ratio as determined by .sup.1H NMR described herein above for the lactic acid monomers over the ethylene oxide monomers.

7.1. Characterization of the Blends

[0118] The melt point was determined as described in Example 1 (1.2.2.), and the intrinsic viscosity was measured with Ubbelohde viscometer (25 C., 0.33 g/dL.). Physical observations were conducted as described in previous examples.

[0119] The results of the blend characterization are reported in Table 19

TABLE-US-00019 TABLE 19 Characterization of the blends Intrinsic Melt range viscosity ID [ C.] [dL/g] Observations Blend A 41-43 0.16 hard-to-semi hard waxy mass Blend C 49-52 0.10 soft waxy mass Blend W 49-51 0.13 soft waxy mass Blend Z 45-47 0.18 sticky moldable creamy mass

7.2. Suitability of the Blends for Putty

[0120] Putties were prepared by mixing the different copolymers with calcium phosphate ceramic granules (1-2 mm) as described in example 3.1. A blind handling test in dry air was conducted on the resulting putties: [0121] Blend A: hard putty, which needs to be warmed up in hands for some minutes to get softness and be shapeable; [0122] Blend C: malleable putty, without need to be warmed; [0123] Blend W: malleable putty, very similar to the one prepared with Blend C; [0124] Blend Z: weak material, that breaks down easily as soon as shaped.

[0125] The malleability of the four putties has been evaluated also in wet conditions, i.e. they were shaped in water: Blend A and Blend Z disrupted quickly losing their malleability and could not retain ceramic granules. Conversely, Blend C and Blend W retained ceramic granules while being shaped, and could be shaped for a longer time.

[0126] The putties have also been evaluated in a dissolution test over a time period. The putties were cut into discs and placed on one extreme of a glass slide. These slides were then placed in tubes filled with 50 mL PBS, which were kept 45 inclined at 37 C. FIG. 8 shows the main endpoints of the dissolution test of the putties. All putties dissolved in an acceptable manner. Blend C and Blend W had faster dissolution leading to quicker putty disruption as compared to Blend A (PEGs with higher molecular weight) and Blend Z (presence of Pluronic P85, which presents hydrophobic poly(propylene glycol) in its structure).

EXAMPLE 8

Effect of L/EO Ratio (i.e. Role of Lactide Content in the Formulation)

[0127] The effect of L/EO ratio on the copolymer physical properties was evaluated. Three formulations, based on Blend C, were prepared with different lactide content as summarized in Table 20. The melt temperature of the three materials has been determined with differential scanning calorimetry (DSC) in the range 25-65 C. with a temperature increase rate of 10 C./min. The results are shown in Table 20 and FIG. 9, the latter showing DSC curves for the three formulations: the decrease of melt temperature with the increase of lactide content is clearly visible. FIG. 10 shows the physical appearance of the formulations according to the lactide content.

TABLE-US-00020 TABLE 20 Characterization of the blends prepared with different lactide content Actual Melt L/EO temp. Observations (see ID Formulation mol ratio [ C.] also FIG. 10) High 70% wt. L-lactide + 0.93 n/a liquid to semi-liquid 7.5% wt. PEG1000 + mass. When shaped, 22.5% wt. PEG2000 it spreads on the surface Middle 15% wt. L-lactide + 0.10 47.1 soft waxy mass that, 21.25% wt. PEG1000 + when shaped, keeps 63.75% wt. PEG2000 the consistency and can be recovered Zero 25% wt PEG1000 + 0 55.4 brittle mass, no 75% wt PEG2000 shaping is possible

[0128] Varying the content of lactide resulted in blends with different physical characteristics. This example demonstrates the relationship between the actual ratio L/EO (i.e. the ratio lactic acid units (hydrophobic block) over ethylene oxide units (hydrophilic block)), the melting point (Tm), the PEG length and the hardness of the putty, as depicted in FIG. 11.