Polymer-clay composite and organoclay

Abstract

The invention relates to a polymer-clay composite material comprising clay nanoparticles and a polymer, and wherein (a) the polymer comprises phosphate and/or phosphonate ligands; or (b) the polymer-clay composite further comprises linker molecules comprising a phosphate or phosphonate ligand, wherein the linker molecules are arranged to be anchored to the polymer. The invention further relates to organoclays, BMP-clay composite material. Uses, treatments, and manufacturer of the material are also provided.

Claims

1. A polymer-clay composite material comprising clay nanoparticles; and a polymer, and wherein (a) the polymer comprises phosphonate ligands; or (b) the polymer-clay composite further comprises linker molecules comprising a phosphonate ligand, wherein the linker molecules are arranged to be anchored to the polymer, wherein the phosphonate ligands comprise or consist of bisphosphonate, wherein the polymer-clay composite comprises between about 0.5% and about 4% polymer (w/v), and wherein the bisphosphonate ligands ionically bond with the clay nanoparticles in an aqueous environment to crosslink the polymer particles and form a hydrogel.

2. The polymer-clay composite material according to claim 1, further comprising water.

3. The polymer-clay composite material according to claim 2, wherein the polymer-clay composite material is in the form of a hydrogel; or wherein the polymer-clay composite material is in the form of a solid suitable for dissolution in water prior to use.

4. The polymer-clay composite material according to claim 1, wherein the linker molecule is anchored to the polymer.

5. The polymer-clay composite material according to claim 1, wherein the clay nanoparticle comprises or consists of layered silicate.

6. The polymer-clay composite material according to claim 1, wherein the clay nanoparticles are synthetic.

7. The polymer-clay composite material according to claim 1, wherein the clay nanoparticles have an aspect ratio of between about 1:5 and about 1:100.

8. The polymer-clay composite material according to claim 1, wherein the clay nanoparticles comprise or consist of synthetic hectorite.

9. The polymer-clay composite material according to claim 1, wherein the polymer clay composite comprises between about 0.5% and about 4% clay nanoparticles (w/v).

10. The polymer-clay composite material according to claim 1, wherein the polymer comprises or consist of a polymer selected from any of the group comprising polyacrylamide, pectin, alginate, carboxymethylcellulose, methylcellulose, PLGA, PEG, polysaccharide, starch, cellulose, chitin, alginate, hyaluronate; protein, collagen, gelatine, casein, albumin, polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), polyetheleneglycol (PEG), polylactic acid (PLA), and polyhydroxy acid (PHA), or combinations thereof.

11. The polymer-clay composite material according to claim 1, wherein the polymer comprises or consists of glycosaminoglycan.

12. The polymer-clay composite material according to claim 11, wherein the glycosaminoglycan comprises or consists of hyaluronan (HA).

13. The polymer-clay composite material according to claim 1, wherein the polymer-clay composite material further comprise an active agent.

14. The polymer-clay composite material according to claim 1, wherein the polymer-clay composite material further comprises a cell.

Description

(1) Embodiments of the invention will now be described in more detail, by way of example only, with reference to the accompanying drawings.

(2) FIG. 1A shows a backbone of hyaluronic acid in which part of carboxylate groups are modified with side groups X. These groups are terminated with bisphosphonates.

(3) FIG. 1B shows one of two types of attachment of bisphosphonate groups to hyaluronan polymeric backbone through a labile disulfide linker (HA-SS-BP derivative).

(4) FIG. 1C shows one of two types of attachment of biophosphonate groups to hyaluronan polymeric backbone through a linker containing more stable chemical bonds (HA-(BP).sub.3).

(5) FIG. 1D shows another type of attachment where several BP groups are linked to one attachment site of hyaluronan. The bisphosphonate-modified hyaluronan and clay (laponite) nanoparticles are two essential components that form a polymer-clay nanocomposite hydrogel through harnessing the laponite-bisphosphonate interactions. Specifically, the anionic bisphosphonate groups serve to crosslink to the cationic edges of the clay nanoparticle to form a strong hydrogel.

(6) FIG. 2 represents an example of the concept of functionalization of clay nanoparticles with useful functionality X basing on interactions between the nanoparticles and a difunctional BP-X linker. When X is a thiol group, thiol-functionalized nanoparticles can be mixed with pyridyldithio-modified hyaluronan (HA-SSPy) afford a chemically (disulphide) cross-linked hydrogel.

(7) FIG. 3 shows another example of the concept of functionalization of clay nanoparticles with useful functionality X. Specifically, thiol-functionalized nanoparticles (X is a thiol group) participate in a UV-light triggered thiol-ene chemical cross-linking by allyl-modified hyaluronan (HA-allyl) affording a thioether cross-linked hydrogel.

(8) FIGS. 4A-4D show SEM images of different types of physical hyaluronan-bisphosphonate⋅laponite hydrogels of the invention. L003 corresponds to chemically (disulfide) cross-linked hydrogel formed through mixing of pyridyldithio-modified hyaluronan (HA-SSPy) with the thiol-functionalized Lanonite NPs. L006 corresponds to physically cross-linked hydrogel prepared from HA-(BP).sub.3 derivative and Lanonite NPs. Preparation of L007 and L008 hydrogels was analogous to the preparation of L006 except that either Lanonite NPs (for L007) or HA-(BP).sub.3 (for L008) were pre-treated with sodium pyrophosphate or magnesium chloride respectively.

(9) FIG. 5A shows thiol-triggered disassembly of hydrogels containing disulfide labile linkages between BP groups and HA backbone upon action of a reducing agent (such as dithiothreitol, DTT).

(10) FIG. 5B shows hydrogels containing thioether linkages, which are stable under the treatment illustrated.

(11) FIGS. 6A-6E show dynamic light scattering (DLS) spectra of nanoparticles obtained after DTT treatment of hydrogel L001 (FIG. 6A), hydrogel L002 (FIG. 6B), and hydrogel L003 (FIG. 6C). For comparison, DLS spectrum of parent Laponite NPs (FIG. 6D) and thiol-functionalized Laponite NPs (FIG. 6E) in water are shown. Hydrogels L001 and L002 were obtained from HA-SS-BP derivative and Lanonite NPs, while hydrogel L003 was obtained chemically between thiol-functionalized Laponite NPs and pyridyldithio-modified hyaluronan (HA-SSPy).

(12) FIG. 7 shows the appearance of natural (non-modified) hyaluronan (left image) and bisphosphonate-modified hyaluronan (middle image) after mixing with a dispersion of laponite nanoparticles. The right image shows that the formed physical hyaluronan-bisphosphonate⋅Laponite hydrogel possesses self-healing properties. Frequency sweep experiment shows that G′ and G″ for the mixture of non-modified hyaluronan and Laponite i.e. represents a liquid in a range of frequencies. Oppositely, G′ is higher than G″ for the mixture of hyaluronan-bisphosphonate and laponite (gel state). The same tendency of physical gel formation was also observed upon interaction of hyaluronan-bisphosphonate with calcium phosphate nanoparticles.

(13) FIGS. 8A-8B show details of loading of different types of hydrogels with a model protein, cytochrome c (cyt c). HA-BP⋅Laponite physical gel was formed by physical interactions between polymeric hyaluronan-bisphosphonate and Laponite nanoparticles. HA-BP chemical gel was formed as a result of hydrazone cross-linking between polymer chains of aldehyde-modified hyaluronan (HA-al) and hydrazide & bisphosphonate dual-modified hyaluronan (HA-by-BP). Control HA chemical gel was formed as a result of hydrazone cross-linking between polymer chains of HA-al and hydrazide-modified hyaluronan (HA-hy). Control Laponite physical gel was formed from the dispersion of laponite nanoparticles. 0.3 mL hydrogels were incubated in 3 mL of PBS containing 3 mg of the protein for 5 days. FIG. 8A shows images of the protein feeding solutions after loading while the lower panel shows images of the hydrogels after loading. The protein loaded hydrogels were then incubated in pure PBS. FIG. 8B shows the amount of the released cyt c over time.

(14) FIGS. 9A-9D provide schematic illustrations of different uses of the invention. In particular, FIG. 9A shows polymer strands linked to each other via a clay nanoparticle interaction with phosphate or phosphonate ligands on the polymer strands. FIG. 9B shows polymer strands linked to each other via a clay particle interaction with phosphate or phosphonate ligands on the polymer strands, wherein * represents a link, such as a disulphide bond between the phosphonate or phosphate ligand and the polymer strand. FIG. 9C shows a functionalised organoclay having active agent X anchored to the clay nanoparticle via a phosphonate or phosphate ligand. FIG. 9D shows a polymer hydrogel functionalised by linking to an organoclay having active agent X anchored to the clay nanoparticle via a phosphonate or phosphate ligand. The clay nanoparticle may or may not be linked to the polymer via phosphate or phosphonate ligand.

(15) FIGS. 10A-10B show clay gels localise BMP2 for enhanced effect in vitro. FIG. 10A shows pre-incubation of BMP2 solutions in the presence of clay gel capsules eliminates the characteristic ALP dose response to BMP2 by C2C12 cells. FIG. 10B shows spotted and dried clay films enhanced ALP activity at doses below that required under standard culture conditions. Enhanced ALP activity was observed when BMP2 was added exogenously to the media but not when premixed with clay. Inset shows enhanced AP activity to be localised to cells growing directly upon clay films. Scale Bar=200 um.

(16) FIGS. 11A-11D show the effect of BMP2 and FCS concentration, cell density and BMP2 preincubation time on ALP activity upon clay films. Clay gel enhancement of ALP is BMP2 dose dependent (FIG. 11A) attenuated by excess fetal calf serum (FIG. 11B) independent of cell density (FIG. 11C) and prolonged over time in association with clay gels. Presence of Laponite film increased ALP activity following incubation of BMP for 2-4 hours prior to cell seeding (FIG. 11D).

(17) FIGS. 12A-12D show BMP2 loaded clay gels functionalise non-viable bone graft to enhance osteogenesis in vitro and in vivo. Pre-coating non viable trabecular bone graft with clay gel enhances the ALP activity in seeded cells in response to BMP2 (bottom) compared to uncoated bone graft (top). Laponite gels localise labelled protein (BSA) within the trabecular structure of bone graft both when premixed with laponite and when added exogenously in PBS. In vivo, enhanced bone formation was observed with bone graft perfused with Laponite with BMP-2 compared to BMP2 alone and bone alone. This was the case whether BMP-2 solution was premixed with Laponite or applied exogenously to Laponite perfused graft at point of implantation.

(18) FIG. 13 shows alternate modes of BMP-2 loading in clay gels induce alternate modes of ectopic ossification. Direct, appositional bone formation was observed upon bone graft surfaces (and enhanced by Laponite gel) in response to exogenously applied BMP-2. This was in contrast to endochondral ossification observed, localised within Laponite gels, in response to premixed BMP-2. LAP=Laponite gel, BG=Bone Graft. Scale bar=50 um.

(19) FIGS. 14A-14D show that only Laponite, not alginate, sustains ectopic bone formation at low doses of BMP2. 500 ng and 40 ng doses of BMP2 were premixed with Laponite or Alginate gels and perfused through a collagen sponge. Both contingency analysis of total numbers (FIG. 14B) and median scaffolds implanted per mouse (FIG. 14C) show increased chance of bone formation in laponite vs. alginate at low but not high dose of BMP2. Significantly greater bone volume per ng BMP was achieved with ‘super low’ doses of BMP2 in Laponite, compared to alginate and Laponite gels with low dose' BMP (FIG. 14D). Ectopic bone formation was observed with Alginate and Laponite containing 7 ng/ul BMP, however at least the lower dose of BMP, bone formation was only seen with Laponite (FIGS. 14A-14C).

METHODS AND MATERIALS

(20) Determination of Interactions Responsible for Cross-Linking Between HA-BP Macromolecules and Laponite Nanoparticles (NPs). HA-BP⋅Laponite Hydrogel Rheology and Interaction Study

(21) Bisphosphonate (BP) groups were linked to hyaluronic acid of molecular weight 150 kDa via either stable thioether linkages (thereafter named as HA-(BP).sub.3) or labile disulfide linkages (thereafter named as HA-SS-BP). Structures of the derivatives are given in FIGS. 1A-1D. Gels were provided according to the following compositions (DS.sub.BP designates degree of substitution with bisphosphonate groups):

Example 1

(22) 6 mg of HA-SS-BP (DS.sub.BP=25%) was dissolved in 150 μL H.sub.2O, while 3 mg Laponite was separately dissolved in 150 μL H.sub.2O. The obtained aqueous solutions were mixed affording a composition of 2% HA and 1% Laponite. This composition was designated as L001.

Example 2

(23) 6 mg of HA-SS-BP (DS=25%) was dissolved in 150 μL H.sub.2O, while 6 mg Laponite was separately dissolved in 150 μL H.sub.2O. The obtained aqueous solutions were mixed affording a composition of 2% HA and 2% Laponite. This composition was designated as L002.

Example 3

(24) Thiol-terminated bisphosphonate derivative (BP-SH in FIG. 2) was prepared also to assess interactions of low molecular weight bisphosphonates with clay nanoparticles. It was expected that interaction of clay nanoparticles with BP-SH should functionalize the nanoparticles with thiol groups. Hyaluronan modified with dithiopyridyl groups (HA-SSPy in FIG. 2) was prepared to figure out about thiol functionalization of the nanoparticles through a simple gel test. For that, 6 mg Laponite was dissolved in 150 μL H.sub.2O and 1.7 mg of HS-BP was added to the obtained nanoparticles solution. The resulting Laponite ⋅BP-SH mixture was stirred for 30 min. Separately, a solution of HA-SSPy (6 mg, DS.sub.SSPy=25%) in 150 μL H.sub.2O was prepared. The obtained aqueous solution of HA-SSPy was mixed with Laponite⋅BP-SH mixture affording a composition of 2% HA and 2% Laponite. This composition was designated as L003 (FIG. 2).

Example 4

(25) Another derivative of HA that is reactive to thiols, HA-allyl, was also prepared (FIG. 3). Analogously to example 3, Laponite⋅BP-SH mixture was prepared by dissolving 6 mg Laponite in 150 μL H.sub.2O and adding 1.7 mg of HS-BP to the solution, followed by stirring for 30 min. After that, a solution of HA-allyl (6 mg, DS.sub.allyl=15%) in 150 μL H.sub.2O was added to the solution of Laponite⋅BP-SH. This composition was designated as L004 (FIG. 3).

Example 5

(26) 0.4% solution of free radical initiator Irgacure 29596 was prepared first. Laponite was dissolved in 150 μL initiator solution and 1.7 mg of HS-BP was added to the obtained nanoparticles solution. The resulting Laponite ⋅BP-SH mixture was stirred for 30 min. Separately, a solution of HA-allyl (6 mg, DS.sub.allyl=15%) in 150 μL initiator solution was prepared. The obtained aqueous solution of HA-allyl was mixed with Laponite⋅BP-SH mixture and then exposed to UV light for 10 minutes (36W UV timer lamp, CNC international BV, Netherlands). This composition was designated as L005 (FIG. 3).

Example 6

(27) 6 mg of HA-(BP).sub.3 (DS.sub.BP=7×3=21%) was dissolved in 150 μL H.sub.2O, while 6 mg Laponite was separately dissolved in 150 μL H.sub.2O. The obtained aqueous solutions were mixed affording a composition of 2% HA and 2% Laponite. This composition was designated as L006.

Example 7

(28) 40 mg Laponite was dissolved in 500 μL H.sub.2O and the nanoparticles solution was pre-incubated with 63.7 mg Na.sub.4P.sub.2O.sub.7.10H.sub.2O in 500 μL H.sub.2O for 1 hour. 150 μL of the above solution containing 6 mg Laponite and 9.555 mg Na.sub.4P.sub.2O.sub.7.10H.sub.2O was then mixed with the solution of HA-(BP).sub.3 (6 mg, DS.sub.BP=7×3=21%) in 150 μL H.sub.2O affording a composition of 2% HA and 2% Laponite. This composition was designated as L007. Assuming molecular weight of Laponite (Na[(Si.sub.8Mg.sub.5.5Li.sub.0.3)O.sub.20(OH).sub.4]) to be 770.75 mg/mmol, 6 mg (6 mg/770.75 mg/mmol=7.79 μmol) of Laponite should contain 7.79 μmol×5.5=42.845 μmol of Mg.sup.2+ ions. This amount of Laponite was pre-treated with 9.555 mg (9.555 mg/446.06 mg/mmol=0.0214 mmol=21.4 μmol) of Na.sub.4P.sub.2O.sub.7.Math.10H.sub.2O. Therefore, the ratio of Mg.sup.2+ in Laponite to P.sub.2O.sub.7.sup.4− was 2:1, i.e. half of magnesium ions could be screened by pyrophosphate ions and thus un-available for interaction with BP groups.

Example 8

(29) Solution of HA-(BP).sub.3 (6 mg, DS=7×3=21%) in 100 μL H.sub.2O was pre-incubated with 18.6 mg MgCl.sub.2 in 50 μL H.sub.2O for 1 hour. During incubation viscosity of the solution was increased indicating coordination of BP groups on HA to soluble Mg.sup.2+ ions. Solution of 6 mg Laponite in 150 μL H.sub.2O was then mixed with the above solution affording a composition of 2% HA and 2% Laponite. This composition was designated as L008. 6 mg of HA-(BP).sub.3 used in the experiment contained 6 mg/400 mg/mmol×0.21=0.00315 mmol of BP groups. Therefore, the ratio of free Mg.sup.2+/BP was 18.6 mg/95.22 mg/mmol/0.00315 mmol=62:1. On the other hand, the ratio of free Mg.sup.2+ to the amount of Mg.sup.2+ in Laponite nanoparticles was 4.55:1.

(30) The formed gels were set for 4 to 6 days and examined by rheology measurements (before swelling). After rheology measurements, the gels were incubated in PBS for another 24 hours. The equilibrated gels were again examined by rheology measurements (after swelling). The rheology measurements are provided in Table 1 below.

(31) TABLE-US-00001 TABLE 1 Before swelling After swelling Mass, G′, G″, Mass, G′, G″, Hydrogel mg Pa Pa mg Pa Pa L001 230.0 526 81 263.4 395.6 36.7 L002 227.4 1064 180 203.8 585 60.0 L003 208.2 1489 34 252.4 1942 88.0 L005* 187.0 1247 86 158.8 912 43.0 L006 237.0 5660 241 269.7 5669 206 L007 232.7 2277 29 273.0 3638 83.5 L008 229.0 6400 618 213.7 4235 396 *Part of the mixture was not cross-linked most probably due to poor light penetration (UV illumination was performed in plastic syringe with only one side opened for direct light exposure). Therefore, the mass of the gel (187 mg) was less than the average mass of other gels (227 mg).
Conclusions for Rheology Study

(32) 1) Use of Laponite with higher concentration (2% vs. 1%) affords stronger gels as exemplified by gels L002 (G′=1064 Pa) and L001 (G′=526 Pa).

(33) 2) Thiol-ene photo-chemical addition of BP-acrylamide to HA-thiol provides an attachment of approximately three BP groups to one thiol group of HA. This results in a brush-like arrangement of BP groups along the HA backbone, as in HA-(BP).sub.3 derivative. Oppositely, disulfide attachment of BP-thiol reagent to HA-SSPy derivative results in tethering of only one BP group to a side chain of the HA backbone, as in HA-SS-BP derivative (FIG. 1C). Moreover, BP groups are attached though more labile disulfide linkages in HA-SS-BP derivative. Eventually, the use of HA-(BP).sub.3 (DS.sub.BP=21%) derivative afforded much stronger gel L006 (G′=5660 Pa) than the use of HA-SS-BP (DS.sub.BP=25%) which yielded gel L002 (G′=1064 Pa).

(34) 3) When Laponite nanoparticles were pre-incubated with pyrophosphate ions prior to mixing with HA-(BP).sub.3, it weakened twice interactions between polymeric HA component and the inorganic nanoparticles (5660 Pa and 2277 Pa for gels L006 and L007 respectively before swelling). After swelling in PBS, the difference in elastic modulus between the gels becomes less (5669 Pa and 3638 Pa for gels L006 and L007 respectively). These observation confirmed participation of Laponite Mg.sup.2+ ions in interaction with HA polymer. Since pyrophosphate is known to interact with Laponite Mg.sup.2+ ions as well as bisphosphonates are analogs of pyrophosphates, it indicates that pyrophosphates most probably displace BPs of HA-(BP).sub.3 from interactions with Mg.sup.2+ ions located on edges of Laponite NPs. Swelling of gel L007 in PBS should cause diffusion of pyrophosphates from the gel, their elimination from competing interactions with BPs, and subsequently to strengthening of the gel.

(35) 4) Oppositely, pre-incubation of HA-(BP).sub.3 with MgCl.sub.2 followed by mixing with Laponite NPs only made the resulting gel L008 stronger (G′=6400 Pa) as compared with gel L006 (G′=5660 Pa). It seems that Mg.sup.2+ ions in solution participates in additional bridging interactions between Laponite NPs and HA-(BP).sub.3 polymer rather than displacing BP groups from interactions with Laponite Mg.sup.2+ ions. Further swelling of gel L008 in PBS eliminated free Mg.sup.2+ ions from the gel and softened the gel (G′=4235 Pa), while almost no change in elastic modulus occurred upon swelling of gel L006 (G′=5669 Pa) which was prepared in pure water.

(36) 5) BP groups on HA polymer are indeed responsible for direct interactions with Laponite NPs and formation of physical gels. This was demonstrated by studying gels L002, L003, and L005. Gel L002 was formed as a result of physical interactions between HA-SS-BP polymer and Laponite NPs. This physical gel can essentially be depicted as HA-SS-BP⋅Laponite. We hypothesized that similar gel structure can be obtained using a chemical thiol-disulfide exchange reaction between polymeric HA-SSPy derivative (FIG. 2) and Laponite⋅BP-SH, assuming that thiol-functionalized Laponite NPs are indeed generated upon interaction between Laponite NPs and thiolated low molecular weight bisphosphonate HS-BP (FIG. 2). Chemical structure of HS-BP is rather simple permitting limited number of options for interactions with Laponite NPs and only interaction through BP side leaves thiol groups free for chemical cross-linking. Moreover, thiol-decorated Laponite NPs should participate in all reactions peculiar to thiols. Therefore, gel L005 was formed upon photo-initiated thiol-ene addition reaction of Laponite⋅BP-SH to allyl-derivatized HA. It is noteworthy that no gel was formed without UV light (composition L004). In general, treatment of Laponite with low molecular weight BPs screens positively charged edges of Laponite NPs excluding them from interactions described by a “playing cards” model of Laponite gel.

(37) Hydrogel Degradation Study

(38) Gels L001, L002, L003, L005, and L006 were divided into two parts:

(39) L001-1 (104.2 mg) and L001-2 (128.3 mg)

(40) L002-1 (96.4 mg) and L002-2 (97.4 mg)

(41) L003-1 (102.2 mg) and L003-2 (93.1 mg)

(42) L005-1 (58.9 mg) and L005-2 (66.9 mg)

(43) L006-1 (120.9 mg) and L006-2 (128.0 mg)

(44) First parts of the gels L001, L002, L003, L005, and L006 as well as gels L007 and L008 were washed by repeated swelling in pure water (3×20 min and 1×16 hours). These samples were analyzed by scanning electron microscopy and images are shown in FIGS. 4A-4D.

(45) Second parts of the gels L001, L002, L003, L005, and L006 were treated with 5 mL of 40 mM dithiothreitol (DTT) for 16 hours. Gels L001-2, L002-2, and L003-2 were dissolved, while gels L005-2 and L006-2 were intact. These results again confirmed that physical gels were formed due to interactions of BP groups on HA polymers with Laponite NPs. Thus, in HA-(BP).sub.3—Laponite gel (L006), the linkage between HA backbone and BP groups cannot be cleaved with DTT. It is also true for the gel formed by photo-initiated thiol-ene cross-linking of Laponite⋅BP-SH nanoparticles with HA-allyl derivative (i.e. L005). The thioester bond that is formed between Laponite NPs and HA macromolecules in this case is insensitive to DTT. However, HA-SS-BP⋅Laponite gels (L001, L002, and L003) have labile disulfide bond between the HA backbone and BP groups of HA polymers. Treatment with DTT can hence disconnect Laponite NPs from HA polymers and thus disassemble the hydrogel in the case of coordination bonding of Laponite NPs to BP groups on HA but not, for example, to HA carboxylate groups (FIGS. 5A-5B).

(46) The dissolved gels L001-2, L002-2, and L003-2 were filtered through a glass wool to remove some remaining visible parts of hydrogels and then examined by DLS (dynamic light scattering).

(47) Digest from L001-2 (FIG. 6A)

(48) Z-Average (d.nm): 158.4 nm

(49) PDI: 0.29

(50) Intercept: 0.949

(51) Result quality: Good

(52) Digest from L002-2 (FIG. 6B)

(53) Z-Average (d.nm): 211.7 nm

(54) PDI: 0.447

(55) Intercept: 0.944

(56) Result quality: Good

(57) Digest from L003-2 (FIG. 6C)

(58) Z-Average (d.nm): 204.5 nm

(59) PDI: 0.352

(60) Intercept: 0.943

(61) Result quality: Good

(62) For comparison, 150 μL of 4% Laponite was diluted with 5 mL water and then examined by DLS:

(63) Laponite NPs in water (FIG. 6D)

(64) Z-Average (d.nm): 60.18 nm

(65) PDI: 0.364

(66) Intercept: 0.950

(67) Result quality: Good

(68) Finally, 6 mg Laponite in 150 μL H.sub.2O was treated with 1.7 mg of HS-BP for 30 min to give Laponite⋅BP-SH (conditions of preparation of gel L003, FIG. 2). Laponite⋅BP-SH was then diluted with 5 mL water and then examined by DLS:

(69) Laponite⋅BP-SH in water (FIG. 6E)

(70) Z-Average (d.nm): 53.65 nm

(71) PDI: 0.218

(72) Intercept: 0.41

(73) Result quality: Good

(74) Conclusions for DLS Study

(75) 1) Design of disulfide linkage between backbone of HA polymer and BP group allowed mild disassembly of the corresponding hydrogels. It was expected to obtain the size of NPs after gel disassembly similar to the size of original Laponite NPs. However, the size of hydrogel-derived NPs was in the range 160-210 nm versus 50-60 nm for original Laponite NPs. Exact calculations revealed only one BP group per 2.5 nanoparticles of Laponite (3.15 μmol of BP groups in 6 mg of HA-(BP).sub.3 and 7.79 μmol of nanoparticles in 6 mg of Laponite, i.e. 7.79/3.15≈2.5). This means that not a single Laponite nanoparticle but rather a cluster of Laponite NPs, associated through electrostatic interactions, can function as a difunctional cross-linker for HA macromolecules. In other words, both inherent electrostatic association of Laponite NPs as well as coordination of bisphosphonated HA polymer to the Laponite associates through BP⋅Mg.sup.2+ coordination may take place during mixing of the organic and inorganic components leading to the formation of physical gel.

(76) 2) Laponite NPs at higher concentrations associate into larger clusters as can be seen from disassembly of gels L001 versus L002.

(77) 3) It is noteworthy that physical [HA-SS-BP+Laponite NPs L002] and chemical [HA-SSPy+Laponite⋅BP-SH.fwdarw.L003] pathways give the hydrogels of the same HA-SS-BP⋅Laponite structure. Disassembly of gels L002 and L003 should give the same Laponite⋅BP-SH NPs which was indeed confirmed by DLS study (211.7 nm and 204.5 nm for Laponite⋅BP-SH NPs derived from gels L002 and L003 respectively).

(78) Cytochrome c (Cyt c) loading and release studies

(79) In this study, the new invented physical hydrogel was compared with its chemical HA analogues either containing or not containing BP groups. For this purpose, several HA derivatives were prepared containing different appended functional groups: aldehyde-modified HA (HA-al), hydrazide-modified HA (HA-hy), and hydrazide and bisphosphonate dually modified HA (HA-BP-hy). The synthesis and structure of all these derivative has been documented by us previously (Xia Yang. et al. (2012) Chemistry of Materials 24, no. 9: 1690-1697). Hydrazone cross-linked hydrogels can be obtained upon mixing of aqueous solutions of HA-al with either HA-hy or HA-BP-hy.

(80) 40 mg of solid Laponite NPs were added under vigorous stirring to 1 mL water and stirring was continued until complete dissolution of the NPs. This afforded 4% Laponite solution.

(81) Four types of hydrogels were prepared:

(82) 1) HA-BP⋅Laponite physical gel by mixing of 6 mg HA-BP in 150 μL H.sub.2O and 6 mg Laponite in 150 μL H.sub.2O.

(83) 2) HA-BP chemical gel by mixing of 3 mg HA-BP-hy in 150 μL H.sub.2O and 3 mg HA-al in 150 μL H.sub.2O.

(84) 3) HA chemical gel by mixing of 3 mg HA-hy in 150 μL H.sub.2O and 3 mg HA-al in 150 μL H.sub.2O.

(85) 4) Laponite physical gel by dissolving 34 mg Laponite in 850 μL H.sub.2O

(86) Gels 1)-3) were formed in three syringe molds and allowed to set for almost 24 hours. Degree of hydrazide modification in HA-BP-hy and HA-hy was the same (10%) which ensured the same cross-linking density on two chemical gels. On the other hand, Amount of HA was also kept the same in all three hydrogel samples (2%). Degree of bisphosphonate modification in HA-BP and HA-BP-hy was the same (8%). Gel 4) was formed in a vial upon standing the 4% Laponite solution overnight.

(87) Mechanical properties of hydrogels after setting.

(88) Hydrogel 1), m(hydrogel)=266 mg, G′=1150 Pa

(89) Hydrogel 2), m(hydrogel)=260 mg, G′=401 Pa

(90) Hydrogel 3), m(hydrogel)=230 mg, G′=1260 Pa

(91) G′ values are shown for frequency 0.5 Hz. Normal force on hydrogels was between 0.015 and 0.02. Chemical HA hydrogel was stronger than chemical HA-BP hydrogel which can be attributed to the repulsive forces between BP groups for HA-BP hydrogel.

(92) Loading Cyt c to Hydrogels 1)-3).

(93) 10 mg of Cyt c was dissolved in 10 mL PBS and 3 mL of the prepared solution was added to each hydrogel sample. The hydrogels were equilibrated in the Cyt c solution for 5 days.

(94) Loading Cyt c to Hydrogel 4).

(95) 3 mg Cyt c/3 mL PBS was added to 277 mg of hydrogel 4). The hydrogel was equilibrated in the CytC solution for 3 days.

(96) Images of hydrogels as well as images of the corresponding Cyt c feeding solutions after loading are shown in FIG. 8A.

(97) Release of Cyt c from Hydrogels.

(98) Hydrogels 1)-4) were placed in 3 mL of PBS after completion of CytC loading by diffusion. At certain intervals of time, PBS medium was withdrawn from the hydrogels and replaced with the fresh one. The collected samples of the release media were later evaluated by UV-Vis spectrophotometry (FIG. 8B).

(99) Clay Nanoparticle Gels Localise and Enhance the Efficacy of BMP Induced Bone Formation

(100) Every year over 2 million people suffer a fracture in the UK alone, while the majority heal uneventfully, in fractures of the lower limb, patients often require 2-4 months off work and in high energy injuries of the tibia up to 40% do not heal. Spinal fusion or arthrodesis is a key treatment in the management of a range of conditions including: scoliosis, degenerative disc disease, spinal stenosis, and trauma. In the last decade rates of spinal fusion in the USA have increased by 137%. Autologous bone grafting (ABG) is considered the gold standard therapy in treatment of fracture non-union, and in mediating spinal fusion. ABG is associated with patient morbidity and volume of graft available is strictly limited. Allograft and synthetic bone products have been developed to replace ABG, however they lack osteogenicity and are less effective in mediating fracture union and arthrodesis than ABG.

(101) Bone Morphogenetic Protein is a growth factor which has been used in clinical practice to replace ABG and stimulate fracture healing and spinal fusion. In clinical practice solubilized BMP is applied as a solution onto a collagen sponge and placed at the fracture or fusion site, around 50% of the BMP is released within 3-6 days, as a consequence relatively large doses are required. Studies have demonstrated significant adverse effects with BMP use such as: Heterotopic ossification, osteolysis, and swelling which were associated with the dose of BMP used. Development of a highly efficient BMP delivery vehicle offers the potential to reduce the effective dose of BMP, facilitating fracture healing and arthrodesis without precipitation of serious adverse effects.

(102) Smectities, are a group of synthetic clays, the unit structure of which consists of two tetrahedral silica sheets sandwiching an octahedral sheet composed of Aluminium or Magnesium. Upon hydration Smectites delaminate to form thixotropic gels, with the charged Smectite sheets giving rise to multiple sites for protein binding. Laponite, has been used in the pharmaceutical industry, and is considered non-toxic.

(103) This study validates the ability of Laponite to localize the activity of exogenously applied BMP in vitro, and enhance the activity of BMP mediated bone formation in vivo.

(104) Clay Gels Localise the Activity of Exogenously Applied BMP In Vitro

(105) The response of C2C12 cells (a myoblastic cell line) to BMP-2 premixed with media or Laponite prior to cell seeding was previously investigated. C2C12 cells cultured on BMP premixed in media demonstrated a characteristic increase in Alkaline Phosphatase (ALP) Activity.sup.13, whilst BMP-2 premixed with Laponite did not (FIG. 10A). In contrast, exogenous application of BMP in the media resulted in localisation of ALP activity to the clay (FIG. 1B). Laponite is known to adsorb proteins. It appears that BMP-2 premixed with Laponite is bound within the clay and unavailable to cells, whilst with exogenous application BMP is localised to the surface of the clay and thus able to stimulate C2C12 cells as observed in FIG. 10B. No effect of Laponite on viability of C2C12 cells or Human Bone Marrow Stromal Cells (HBMC) was observed. In contrast to previous work.sup.14,15 no intrinsic osteogenic effect of Laponite on HBMC was identified. This discrepancy is likely to be due to variation in Laponite preparations employed, Wang.sup.14 utilised sintered Laponite whilst Gaharwar.sup.15 applied Laponite to a solution in contrast to dry Laponite films in the present study.

(106) Clay Gels Enhance and Prolong Activity of BMP In Vitro

(107) Presence of clay was observed to enhance the cellular response to increasing concentrations of exogenous BMP (FIG. 11A). A differential cellular response to Fetal Bovine Serum (FBS) was observed on Laponite, with ALP activity peaking at 2% and 5% FBS for Laponite and tissue culture plastic (TCP) respectively. Laponite has been shown to exhibit preferential protein binding.sup.16, it is postulated that the different effect of FBS in presence of Laponite may result from displacement of Laponite bound BMP.

(108) In order to define if increased ALP activity on Laponite films resulted from an effect of cell density or activity per cell the response of ALP activity to cell seeding density on Laponite and TCP was characterised. ALP activity was proportional to cell seeding density on both Laponite and TCP, however, through the range tested cell density and ALP activity per cell was greater on Laponite (FIG. 11C). This demonstrated ALP activity per cell was increased, with reduced cell density on Laponite likely to be secondary to BMP mediated stimulation of differentiation at the expense of proliferation. Presence of Laponite film increased ALP activity following incubation of BMP for 2-4 hours prior to cell seeding (FIG. 11D). The demonstrated increased ALP activity in the presence of Laponite could be either secondary to modulated BMP activity, or an effect of Laponite on BMP localisation, in the context of previous work.sup.12 the latter is more likely.

(109) Clay Bound BMP Enhances Allograft Bone Formation

(110) Laponite gel was observed to maintain BMP and labelled Bovine Serum Albumin (BSA) to allograft despite undergoing a saline wash (FIGS. 12A and 12B). In the absence of Laponite BMP and BSA were readily displaced from allograft during washing. The ability of Laponite gel to enhance bone formation on acellular allograft mediated by 1 ug BMP per implant in a murine mode was subsequently investigated. Allograft cylinders were implanted subcutaneously in nude mice and loaded with: (i) BMP, (ii) Laponite gel, (iii) Laponite with premixed BMP, (iv) Laponite and exogenous BMP, or (v) left blank. MicroCT performed prior to implantation and at 28 days demonstrated that increase in bone volume was significantly greater with BMP applied in the presence of Laponite, compared to BMP with allograft (FIG. 12C).

(111) Application of BMP to allograft resulted in reduction of increase in bone volume compared to allograft alone. In addition to stimulating osteogenesis, BMP is also known to stimulate osteolysis.sup.17 and it is postulated the relative effect upon these opposite processes is dependent on magnitude and rate of BMP delivery. Histological analysis failed to show any new bone on allograft in the absence of BMP. Whilst some new bone was observed with allograft and BMP, more areas of new bone formation were evident in the presence of BMP and Laponite (FIG. 12A-12D). Modulation of BMP release by the Laponite may result in a predominately osteogenic response as opposed to osteolytic when BMP is delivered in isolation. In concordance with the in vitro work, Laponite alone, was not seen to have an osteogenic effect. There was no significant difference of increase in allograft bone volume with Laponite and premixed BMP-2 or Laponite and exogenous application of BMP-2 on micro CT. However, on histological analysis exogenous BMP appeared to promote appositional bone formation, whereas endochondral bone formation predominated when BMP-2 was premixed with the Laponite gel prior to application (FIG. 13).

(112) The in vitro results suggest premixing results in BMP localisation within the gel, if this replicated in vivo only osteoprogenitor cells present within the gel may be activated by the BMP, in contrast to exogenous BMP which is available to stimulate cells on the surface of the gel. The difference in biomechanical and biological environments within the gel and on the gel surface may explain the stimulation of endochrondral and appositional osteogenesis observed in Laponite with premixed and exogenously applied BMP, respectively.

(113) Clay Gels Reduce the Dose of BMP Required for Ectopic Bone Formation

(114) It was next investigated if Laponite gel was able to reduce the dose of BMP required to stimulate bone formation. BMP was mixed in Laponite and Alginate to produce gels containing 7 ng/ul and 0.57 ng/ul BMP. Gels were absorbed by collagen sponge cylinders and implanted subcutaneously in MF-1 mice. Bone volume was assessed fortnightly, and at 8 weeks histological analysis was performed. It was chosen to deviate from clinical method of BMP delivery by using Alginate gel and collagen in lieu of collagen alone as this enabled comparison of Laponite with another hydrogel, rather than water. Alginate has a proven track record in growth factor delivery.sup.18 and has been shown to mediate BMP delivery more efficiently than collagen alone.sup.19. Ectopic bone formation was observed with Alginate and Laponite containing 7 ng/ul BMP, however at the lower dose of BMP, bone formation was only seen with Laponite (FIGS. 14A-14C).

(115) Bone volume formed per ug BMP was significantly greater with Laponite and low dose BMP compared to alginate with high or low dose BMP (FIG. 14D). Volume of gel loaded onto individual collagen cylinders was recorded, and bone volume produced per unit BMP expressed graphically (FIG. 14D). Bone volume was directly proportional to BMP with Laponite and low dose BMP, while no correlation was seen with gels and high dose BMP. These results are suggestive that the in vivo response to BMP was saturated with gels containing high dose BMP, this observation is supported in the literature; Boerckel.sup.19 reported a dose dependent increase in bone volume as BMP was increased from 6.37 to 31.83 ug/cm.sup.3 at a rat femoral defect, and Peleaz.sup.20 found that stimulation of bone formation in a rat calvarial defect was saturated around 25-50 ug/cm.sup.3. Inter-study comparison of BMP dosing is inherently challenging due to variation in BMP preparation and the plethora of species and in vivo models employed.sup.18. To facilitate comparison of BMP doses used in this study BMP doses are expressed as ug BMP per volume of defect (cm.sup.3) from some key publications (table 1). This study, in which Laponite hydrogel was used as a delivery vehicle, demonstrates the lowest recorded BMP dose to stimulate ectopic bone formation, an environment which is considerably less osteogenic than orthotopic, or spinal fusion models such as employed by Lee.sup.21, in which the defect is adjacent to bleeding bone.

(116) TABLE-US-00002 TABLE 1 Defect Dose Min. effect volume BMP dose BMP Author BMP carrier Species Model (uL) (ug/cm.sup.3) (ug/cm.sup.3) Lee Heparin based rat Posterio-lateral 200 0.5 0.5 2015.sup.21 hydrogel spinal fusion Heparin based mouse Ectopic 20 50   50 hydrogel (muscle) Gibbs ACS/ mouse Ectopic 63 0.57-6.97  0.57 2015 Laponite (subcutaneous) Boerckel PCL mesh + rat Femoral defect 157 0.64-31.83 6.37 2011.sup.19 Alginate ACS rat Femoral defect 157 0.63-15.91 6.37 Wang none rat Ectopic 50 9.2 9.2 1990.sup.22 (subcutaneous) Pelaez ACS rat calvarial 50 24.87-397.89 24.87 2014.sup.20 Ben- PEG/fibrinogen Nude Ectopic 30 33.95 33.95 David hydrogel mice (subcutaneous) 2013.sup.23 Govender ACS human Tibial fracture 750-1500 750 2002.sup.5 ACS, Absorbable Collagen Sponge. PCL, Polycaprolactone. PEG, Polyethylene Glycol

(117) The ability of Laponite to localise and enhance BMP activity in vitro was shown, while the in vivo study demonstrated that BMP delivered by Laponite stimulated ectopic bone formation at BMP doses approximately 3000 fold smaller than those employed in clinical practice. There is exciting potential of Laponite to safely harness the powerful osteogenic effect of BMP thus facilitating treatment of thousands of patients suffering from non-union of fractures or spinal arthrodesis.

(118) Methods

(119) Laponite Preparation

(120) Laponite gel was prepared as described previously.sup.12. Briefly, Laponite XLG powder was dissolved in distilled water to required concentration % weight Laponite per unit volume. Laponite gel was subsequently sterilised by autoclave and evaporated water replaced. To produce dry Laponite films for cell culture 5 ul of 1% Laponite was placed on TCP and permitted to dry for 2 hours at room temperature prior to cell seeding.

(121) Cell Culture and Analysis of ALP Activity

(122) Unless stated otherwise C2C12 cells were seeded at 1×10.sup.5/cm.sup.2 and cultured with D-MEM containing 1% Penicillin/Streptomycin, 10% FBS, and when present BMP, at 200 ng/ml. Following cell culture for 72 hours, cells were fixed in ethanol and alkaline phosphatase staining performed according to a standard protocol. Representative images were taken using Axiovert 200 microscope and Axiovision software V4.0. Cell Profiler software was used to calculate cell density and ALP staining intensity relative to Laponite or TCP according to surface used for cell culture.

(123) Allograft Preparation

(124) Donated human femoral heads were received from Southampton General Hospital with ethical approval. Cylinders of trabecular bone 4 mm in diameter were removed using a trephine. Samples were cut to remove any subchondral bone to form cylinders 4 mm in length. Sections of trabecular bone approximately 10×10×2 mm were also cut with a bone saw from a second femoral head. Cylinders and bone sections underwent multiple washes in 5% Hydrogen Peroxide and saline to remove cells and fat.

(125) In Vitro Allograft Studies

(126) 20 ul of 1% Laponite gel was applied to a section of acellular allograft and left to dry for 2 hours at 37° C. This allograft section, and a second allograft section which had not received Laponite, were placed in petri dishes. Media containing C2C12 cells and BMP was added and microscopy performed as described above (FIG. 12A). Allograft sections were perfused with: 19 ul Phosphate Buffered Saline (PBS)+1 ul Fluorescein labelled Bovine Serum Albumin (FITC-BSA), 20 ul 2.5% Laponite, 19 ul 2.5% Laponite+1 ul FITC-BSA, premixed with Laponite or applied exogenously following application of Laponite gel to allograft sections. Allograft sections were washed in PBS for 2 minutes, and representative images taken (FIG. 12B).

(127) In Vivo Study of Bone Formation on Allograft

(128) In compliance with ethical approval nude mice were anaesthetised with an intra-peritoneal injection of a midazolam/fentanyl mix. A midline dorsal incision was made, 3 allograft cylinders were implanted on each side and wounds closed with clips. Immediately prior to implantation cylinders were perfused with: 1) 20 ul PBS+1 ug BMP, 2) 20 ul 2.5% Laponite, 3) 20 ul 2.5% Laponite+1 ug BMP mixed in prior to application, 4) 20 ul 2.5% Laponite with 1 ug BMP added subsequently or 5) left blank as a control. Five mice were used in total, with n=6 for each of the 5 groups.

(129) In Vivo Study of Bone Formation in Collagen

(130) MF-1 mice were used, surgery and anaesthesia was performed as above. A collagen sheet 4 mm in thickness was obtained from Medtronic. From this identical cylinders of 4 mm in diameter were prepared using a skin biopsy punch in a sterile environment. BMP solution of 1 ug/ul was added to 2% Laponite and 2% Alginate to produce gels containing 7 ug/ml and 0.57 ug/ml BMP. 140 ul of the gels was transferred to individual wells of a 96 well plate. Sponge cylinders were compressed and allowed to expand while submerged in the gel filled wells. Each mouse received 3 collagen cylinders containing high dose BMP gels on the left side and 3 with low dose BMP gels on the right, one mouse received 6 blank collagen cylinders as a control. In total 17 mice were used, with n=24 for each of the 4 groups: 1) Laponite 7 ug/ml BMP 2) Laponite 0.57 uh/ml BMP 3) Alginate 7 ug/ml BMP 4) Alginate 0.57 uh/ml BMP and n=6 in group 5) collagen only. Gels were made fresh for each individual mouse during induction of anaesthesia, with BMP kept on dry ice until use. Volume of gel remaining following absorption was recorded for each individual cylinder.

(131) Micro CT

(132) All CT scans were performed using Brunker Skyscan 1176, images were reconstructed using NRecon, and analysed using CTAn software. Allograft cylinders were scanned prior to implantation with 50 kV voltage, 500 uA current, 0.5 mm Al filter and a pixel size of 9 um. Following implantation allograft cylinders were removed and scanned again using the same settings. The same scan settings were used during the study of bone formation within the collagen cylinders with the exception that the pixel size was increased to 18 um.

(133) Histology

(134) Allograft samples underwent decalcification in Histoline for 24 hours, collagen samples did not undergo decalcification. Subsequently samples were dehydrated, embedded in wax and sectioned at 9 um thickness. Alcian blue and Sirius red staining was performed according to standard protocols.

(135) Statistical Analysis

(136) Statistical analysis was performed using GraphPad Prism 6.0. Unpaired t-tests were used to compare ALP activity on Laponite with TCP with statistical significance determined using the Holm-Sidak method when BMP or cell seeding density were variables. For BMP incubation study 2-way ANOVA test was performed with P values adjusted to account for multiple comparisons. Fisher's exact test was used to compare number collagen scaffolds demonstrating bone formation. One-way ANOVA test was used to compare mean bone volume formed on collagen and allograft scaffolds.

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