Heparan sulphate which binds BMP2
10086044 ยท 2018-10-02
Assignee
Inventors
- Simon McKenzie Cool (Singapore, SG)
- Victor Nurcombe (Singapore, SG)
- Christian Dombrowski (Singapore, SG)
Cpc classification
A61K2035/124
HUMAN NECESSITIES
C07K14/51
CHEMISTRY; METALLURGY
C08B37/0075
CHEMISTRY; METALLURGY
A61K38/1875
HUMAN NECESSITIES
A61P17/02
HUMAN NECESSITIES
A61P19/08
HUMAN NECESSITIES
A61K31/737
HUMAN NECESSITIES
A61L27/3834
HUMAN NECESSITIES
A61P43/00
HUMAN NECESSITIES
A61L2430/02
HUMAN NECESSITIES
C08B37/0003
CHEMISTRY; METALLURGY
C12N5/0663
CHEMISTRY; METALLURGY
A61L2300/236
HUMAN NECESSITIES
C12N2501/155
CHEMISTRY; METALLURGY
A61K35/28
HUMAN NECESSITIES
A61L2300/252
HUMAN NECESSITIES
B01D15/3823
PERFORMING OPERATIONS; TRANSPORTING
International classification
A61L27/36
HUMAN NECESSITIES
A61L27/54
HUMAN NECESSITIES
C08B37/00
CHEMISTRY; METALLURGY
A61K31/737
HUMAN NECESSITIES
A61K35/28
HUMAN NECESSITIES
Abstract
The invention relates to heparan sulphate GAGs obtained by affinity chromatography using the heparin-binding domain of BMP2. The GAGs were obtained from osteoblast extracellular matrix and from a commercially available heparan sulfate (Celsus HS).
Claims
1. A composition comprising a solid or solidified scaffold coated or impregnated with a therapeutically effective amount of an isolated or substantially purified heparan sulphate composition comprising a heparan sulphate component, wherein the heparan sulphate component is at least 97% HS/BMP2 which is capable of specific binding to SEQ ID NO:1 or 6, and to BMP2, wherein the heparan sulphate composition is obtained by a method comprising: (i) providing a solid support having polypeptide molecules adhered to the support, wherein the polypeptide consists of the amino acid sequence QAKHKQRKRLKSSCKRHP [SEQ ID NO:1]or QAKHKQRKRLKSSCKRH [SEQ ID NO:6]; (ii) contacting the polypeptide molecules with a heparan sulphate fraction obtained from mammalian tissue or extracellular matrix such that polypeptide-heparan sulphate complexes are allowed to form; (iii) partitioning polypeptide-heparan sulphate complexes from the remainder of the mixture; (iv) dissociating the heparan sulphate from the polypeptide-heparan sulphate complexes by disrupting the polypeptide-heparan sulphate complexes; and (v) collecting the dissociated heparan sulphate, wherein the composition is capable of improving bone fracture repair in a mammal compared to a corresponding untreated fracture.
2. The composition of claim 1 wherein the scaffold is further coated or impregnated with BMP2 protein, mesenchymal stem cells, or a combination thereof.
3. The composition of claim 1, wherein the scaffold is a gel.
4. The composition of claim 1, wherein the scaffold is a hydrogel.
5. The composition of claim 1, wherein the scaffold comprises ceramic.
6. The composition of claim 1, wherein the scaffold comprises -tricalcium phosphate (TCP).
7. The composition of claim 1, wherein the scaffold comprises hydroxyapatite (HA).
8. The composition of claim 1, wherein the scaffold comprises hyaluronic acid.
9. The composition of claim 1, wherein the scaffold comprises demineralized bone matrix.
10. The composition of claim 1, wherein the scaffold comprises calcium sulfate.
11. The composition of claim 1, wherein the scaffold comprises collagen.
12. The composition of claim 1, wherein the scaffold comprises fibrin.
13. The composition of claim 1, wherein the scaffold is an allograft.
14. The composition of claim 1, wherein the scaffold is an autograft.
15. The composition of claim 4, wherein the hydrogel comprises carboxymethyl cellulose.
16. The composition according to claim 1 which binds to SEQ ID NO:1 with a K.sub.D of less than 1 M.
17. The composition according to claim 1 wherein the HS/BMP2 is N-sulphated.
18. The composition according to claim 1 wherein the HS/BMP2 is 6-O-sulphated.
19. The composition according to claim 1 additionally comprising BMP2 protein.
Description
BRIEF DESCRIPTION OF THE FIGURES
(1) Embodiments and experiments illustrating the principles of the invention will now be discussed with reference to the accompanying figures in which:
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DETAILED DESCRIPTION OF THE INVENTION
(76) The details of one or more embodiments of the invention are set forth in the accompanying description below including specific details of the best mode contemplated by the inventors for carrying out the invention, by way of example. It will be apparent to one skilled in the art that the present invention may be practiced without limitation to these specific details.
(77) We investigated the potential of GAGs to augment the activities of bone morphogenic protein 2 (BMP2). The highly osteoinductive activity of BMP2 for the murine myogenic cell line C2C12 have been well characterised. Studies both in this cell line, and in vivo, have implicated a role for glycosaminoglycans in modulating this activity.
(78) BMP2's affinity for heparin has similarly been well characterised. Numerous studies have been conducted that have sought to examine the dynamic interaction between BMP2 and GAGs. Some have proposed that the interaction is inhibitory, and so responsible for either sequestering the cytokine away from the receptor or inducing its association with its numerous inhibitors, such as noggin, that have been shown, similarly, to have an affinity for heparin. Alternative findings implicate the interaction between BMP2 and GAGs is one of maintaining a local concentration of the cytokine around cells that require its signalling in order to differentiate into the osteoblast lineage.
(79) These findings also suggest that the association serves to significantly lengthen the half-life of the homodimer, so allowing it to remain active in the ECM for longer periods. As is the case with most systems, the actual role of this interaction is likely to be blend of some, or all of the above.
(80) Although many studies have provided evidence for the interaction that BMP2 has with model sugars, the specific interaction between the BMP2 heparin-binding peptide (BMP2-HBP), a string of amino acids (QAKHKQRKRLKSSCKRHP [SEQ ID NO: 1]) located at the N-terminal end of each BMP2 monomer, and appropriate glycosaminoglycans has received relatively little attention. A major question that arises is whether there is a complementary saccharide sequence embedded within an HS chain that controls the association with an absolute, or at least relative, specificity.
(81) We sought to isolate a sequence-specific glycosaminoglycan that could modulate BMP2 activity via a direct interaction with the cytokine.
EXAMPLE 1
(82) Materials and Methods
(83) Buffer Preparation
(84) Preparation of all buffers for GAG extraction and analysis is conducted with strict attention paid to quality. It is vital that the pH of buffers is maintained at the correct level and that all buffers be filtered and degassed in order to prevent the clogging of columns with precipitates or bubbles. The formation of bubbles, in particular, can cause serious damage to columns, and in the case of sealed, pre-fabricated columns, leads to them becoming unusable.
(85) All buffers used were filtered with 1 PBS without Ca.sup.2+ or Mg.sup.2+ (150 mM NaCl), or double distilled (ddH.sub.2O) to make the final solutions.
(86) Disruption Buffer
(87) The 8M Urea/CHAPS disruption buffer consisted of PBS (150 mM NaCl) with 1% CHAPS, 8M Urea and 0.02% NaN.sub.3 to prevent contamination by microbial growth during storage. This solution was used to disrupt matrix (MX) samples, so was not degassed or filtered.
(88) PGAG Anion Exchange Low Salt (250 mM) Buffer
(89) Low salt PGAG anion exchange buffer comprised PBS (150 mM NaCl) with an additional 100 mM NaCl. The buffer was equilibrated to pH 7.3 with NaOH and 0.02% NaN.sub.3. The solution was then degassed under negative pressure and constant stirring until no further bubbles were released before being filtered through a 0.4 m filter.
(90) PGAG Anion Exchange High Salt (1M) Buffer
(91) High salt PGAG anion exchange buffer comprised PBS (150 mM NaCl) with an additional 850 mM NaCl. The buffer was equilibrated to pH 7.3 with NaOH and 0.02% NaN.sub.3 added. The solution was then degassed under negative pressure and constant stirring before being filtered through a 0.4 m filter.
(92) Pronase/Neuraminidase PGAG Reconstitution Buffer
(93) This buffer was used to reconstitute desalted, PGAG samples after anion exchange in order to prepare them for enzymatic digestion of the associated core proteins. It consisted of 25 mM sodium acetate (CH.sub.3COOHNa). The buffer was equilibrated to pH 5.0 with glacial acetic acid (CH.sub.3COOH). Both pronase and neuraminidase enzymes were reconstituted according to the manufacturer's instructions.
(94) GAG Affinity Chromatography Low Salt (150 mM) Buffer
(95) Low salt GAG anion exchange buffer was made using PBS (150 mM NaCl) without any additional salt. The buffer was equilibrated to pH 7.3 with NaOH and 0.02% NaN.sub.3. The solution was degassed under negative pressure and constant stirring until no further bubbles were released before being filtered through a 0.4 m filter.
(96) GAG Affinity Chromatography High Salt (1M) Buffer
(97) High salt GAG anion exchange buffer was made using PBS (150 mM NaCl) with an additional 850 mM NaCl. The buffer was equilibrated to pH 7.3 with NaOH and 0.02% NaN.sub.3 was added, the solution was then degassed and filtered through a 0.4 m filter.
(98) Desalting Solution
(99) The desalting solution was made using ddH.sub.2O that was equilibrated to pH 7.0 with 0.02% NaN.sub.3. The solution was then degassed and filtered.
(100) Sample Preparation
(101) Matrix samples were disrupted using Disruption Buffer (8M Urea/CHAPS), then scraped off the culture surface in this buffer and stirred overnight at 3.7 C. to ensure maximal lysis. The samples were then centrifuged at 5000 g for 30 min and the supernatant was clarified through a 0.4 m filter in preparation for PGAG extraction via anion exchange chromatography.
(102) Column Preparation & Usage
(103) The choice and preparation of the types of columns to be used for each sequential step in the isolation and characterisation of GAGs is of major importance for the success of the protocol. It was vital that at each step the columns were equilibrated and cleaned with great care.
(104) Anion Exchange Columns
(105) Due to the relatively large quantities of MX substrate used for GAG extraction, and the high load this places on the column system, it was necessary to pack and prepare a large anion exchange column manually, specifically for this study. Capto Q anion exchange beads (Pharmacia) were packed into a Pharmacia XK 26 column (Pharmacia) to produce a column with a maximum loading capacity of 500 ml of MX lysate per run.
(106) Prior to use, both the column and all buffers were equilibrated to room temperature for 30 min, before washing and equilibrating the column in PGAG Anion Exchange Low Salt (250 mM) Buffer for 30 min until all absorbance channels remained stable. The clarified cell lysate was then passed through the column which was again rinsed in 500 ml of low salt buffer to remove any nonspecifically bound debris. PGAGs were then eluted using 250 ml of PGAG Anion Exchange High Salt (1M) Buffer and lyophilised prior to desalting. The column was then rinsed in low salt buffer and returned to 4 C. for storage.
(107) Desalting Protocol
(108) After PGAG/GAG isolation it was necessary to remove the high amount of salt that accumulated in the sample during elution from the column. For this step, all eluted samples of the same experimental group were combined and loaded onto 4 Pharmacia HiPrep 26/10 desalting columns. Prior to use, both the columns and all solutions were equilibrated to room temperature for 30 min before washing and equilibrating the column in Desalting Solution for 30 min until all absorbance channels achieved stability. Lyophilised samples were reconstituted in Desalting Solution, in the minimum possible volume that resulted in a clear solution. This combinatron of columns permitted the loading of up to 60 ml of sample. Those fractions eluting from the column first were lyophilised and retained for further separation or cell culture application. The columns were then rinsed in Desalting Solution and returned to 4 C. for storage.
(109) BMP2-HBP Column Preparation
(110) The isolation of GAGs carrying relative affinities for BMP2 was conducted using a BMP2-HBP column. Approximately 2 mg of biotinylated BMP2-HBP was prepared in 1 ml of the GAG Affinity Chromatography Low Salt (150 mM) Buffer. This amount was loaded onto a HiTrap Streptavidin HP column (Pharmacia) and allowed to attach to the column for 5 min. The column was then subjected to a complete run cycle in the absence of GAGs. The column was washed in 13 ml of low salt buffer at a flow rate of 0.5 ml/min before being subjected to 10 ml of GAG High salt buffer at 1 ml/min. Finally the column was rinsed with 10 ml of low salt buffer. During this process data was carefully monitored to ensure that no peptide elution or column degradation was observed.
(111) GAG+ Sample Isolation
(112) Once the BMP2-HBP column had been prepared and tested for stability under normal running conditions, it was ready to be used for the separation of GAG+ chains from tGAG (total GAG) samples. tGAG samples (6 mg) were prepared in 3 ml of GAG affinity low salt (150 mM) buffer and injected into a static loop for loading onto the column. Prior to use both the BMP2-HBP column and all buffers were equilibrated to room temperature for 30 min before washing and equilibrating the column in low salt buffer for 30 min until all absorbance channels were stable. The sample was then loaded onto the column at 0.5 ml/min and the column and the sample rinsed in 10 ml of low salt buffer at 0.5 ml/min. Retained GAG+ samples were subsequently recovered by elution with 10 ml of high salt (1 M) buffer and lyophilised for desalting. The column was then rinsed in 10 ml of low salt buffer and stored at 4 C.
(113) Pronase/Neuraminidase Treatment
(114) In order to isolate GAG chains from their core proteins, they were digested using pronase and neuraminidase. Lyophilized PGAG samples were resuspended in a minimum volume of 25 mM sodium acetate (pH 5.0) and clarified by filtration through a 0.4 m syringe filter. Total sample volume was dispensed, into 10 ml glass tubes in 500 l aliquots. 500 l of 1 mg/ml neuraminidase was added and incubated for 4 h at 37 C. After incubation 5 ml of 100 mM Tris-acetate (pH 8.0) was added to each sample. An additional 1.2 ml of 10 mg/ml pronase, reconstituted in 500 mM Tris-acetate, 50 mM calcium acetate (pH 8.0), was added to each sample and incubated for 24 h at 36 C. After treatment all volumes were combined and prepared for anion exchange processing by centrifugation and filtration.
(115) GAG Digestion Protocols
(116) The analysis of GAGs, including their sulfated domain sizes and relative sulfation levels, was carried out by using established protocols including degradation by either nitrous acid or lyases.
(117) Nitrous Acid Digestion
(118) Nitrous acid-based depolymerisation of heparan sulfate leads to the eventual degradation of the carbohydrate chain into its individual disaccharide components when taken to completion. Nitrous acid was prepared by chilling 250 l of 0.5 M H.sub.2SO.sub.4 and 0.5 M Ba(NO.sub.2).sub.2 separately on ice for 15 min. After cooling, the Ba(NO.sub.2).sub.2 was combined with the H.sub.2SO.sub.4 and vortexed before being centrifuged to remove the barium sulfate precipitate. 125 l of HNO.sub.2 was added to GAG samples resuspended in 20 l of H.sub.2O, and vortexed before being incubated for 15 min at 25 C. with occasional mixing. After incubation, 1 M Na.sub.2CO.sub.3 was added to the sample to bring it to pH 6. Next, 100 l of 0.25 M NaBH.sub.4 in 0.1 M NaOH was added to the sample and the mixture was heated to 50 C. for 20 min. The mixture was then cooled to 25 C. and acidified with glacial acetic acid to pH 3 in the fume hood. The mixture was then neutralised with 10 M NaOH and the volume was then decreased by freeze drying. The final samples were run on a Bio-Gel P-2 column to separate di- and tetrasaccharides to verify degradation.
(119) Heparinase III Digestion
(120) Heparinase III is an enzyme that cleaves sugar chains at glucuronidic linkages. The series of heparinase enzymes (I, II and III) each display relatively specific activity by depolymerising certain heparan sulfate sequences at particular sulfation recognition sites. Heparinase I cleaves HS chains within NS regions along the chain. This leads to the disruption of the sulfated domains that are thought to carry most of the biological activity of HS. Heparinase III depolymerises HS within the NA domains, resulting in the separation of the carbohydrate chain into individual sulfated domains. Lastly, Heparinase II primarily cleaves in the NA/NS shoulder domains of HS chains, where varying sulfation patterns are found.
(121) In order to isolate potential active domains we focused on the depolymerisation of GAG+ NA regions. Both the enzyme and lyophilised HS samples were prepared in a buffer containing 20 mM Tris-HCl, 0.1 mg/ml BSA and 4 mM CaCl.sub.2 at pH 7.5. The concentration of heparinase III added to each sample is governed by the relative quantity of HS components in the sample. Our analysis, via nitrous acid depolymerisation, indicated that the GAG+ samples consisted of predominantly HS; thus the enzyme was used at 5 mU per 1 g of HS. The sample was incubated at 37 C. for 16 h before the reaction was stopped by heating to 70 C. for 5 min. The sample was then applied to the appropriate column system for further analysis.
(122) Cell Culture
(123) GAG Production
(124) In order to isolate GAG species representative of developing osteoblasts, MC3T3 cells were grown in osteogenic conditions for 8 days. The cellular component was removed via incubation in a dilute solution of 0.02 M ammonium hydroxide (NH.sub.4OH) at 25 C. for 5 min. After 5 min, NH.sub.4OH was removed by inversion of the culture surfaces. Treated cultures were allowed to dry in a laminar flow cabinet overnight. The following day the treated cultures were washed three times with sterile PBS and allowed to dry in the laminar flow cabinet. Prepared matrix cultures were then stored under sterile conditions in 4 C. until primary proteoglycans were liberated via treatment with disruption buffer and anion exchange chromatography.
(125) BMP2-Specific GAG Bioactivity
(126) C2C12 myoblasts were subcultured every 48 h, to a maximum of 15 passages, by plating at 1.310.sup.4 cells/cm.sup.2 in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FCS. Osteogenic differentiation was induced at 210.sup.4 cells/cm.sup.2 in DMEM supplemented with 5% FCS, nominated concentrations of recombinant human bone morphogenic protein-2 (rhBMP2) and glycosaminoglycan fractions with a positive or negative affinity for rhBMP2 (GAG+ and GAG respectively). rhBMP2 and GAG fractions were pre-incubated for 30 min at 25 C. prior to addition to their corresponding C2C12 cultures. The cultures were permitted to grow under these conditions for 5 days, with media for each condition being changed every 48 h, before mRNA samples were extracted and prepared for RQ-PCR analysis. Real time PCR for osteocalcin expression was conduced using the ABI Prism 7000 sequence detection system (PerkinElmer Life Sciences). Primers and probes were designed using Primer Express software (v2.1, PE Applied Biosystems). The target probe was redesigned to incorporate LNA bases and labelled with BHQ-1 (Sigma-Proligo). The ribosomal subunit gene 18S (VIC/TAMRA) was used as an endogenous control, with each condition consisting of three repeats, each tested in triplicate. The raw PCR data was analysed using the ABI Sequence Detector software. Target gene expression values were normalised to 18S expression prior to the calculation of relative expression units (REUs).
(127) Results
(128) Anion Exchange Chromatography
(129) In order to successfully extract GAGs from MX samples, it is necessary to remove other matrix proteins that may contaminate the sample. As GAGs constitute the most negatively charged molecules in the ECM, this is most effectively accomplished with anion exchange chromatography.
(130) Samples were disrupted using 8M Urea/CHAPS buffer and loaded onto the anion exchange column. Unwanted protein and ECM debris were washed from the column and the negatively charged GAGs eluted with 1 M NaCl. A typical chromatogram (
(131) Desalting
(132) Virtually all chromatography methods employed to purify and analyse GAGs at various stages of processing require elution with high-salt buffers. As high salt conditions interfere with affinity-based chromatography, it is necessary to desalt samples after each stage of processing. This process is generally completed with size exclusion chromatography. Under these conditions larger molecules, such as GAGs, exit the column before small molecules, including the salt and small GAG debris. The separation of GAGs from the contaminating salt can be followed on the resulting chromatogram (
(133) BMP2-HBP Column System
(134) Column Preparation
(135) Due to the prohibitive costs involved in creating a BMP2 growth factor column with commercially available reagents, we instead utilised a biotinylated preparation of the known heparin-binding domain of BMP2 (BMP2-HBP). This peptide was immobilised on a Hi-Trap Streptavidin HP column (1 ml) in order to specifically retain GAG chains with an affinity for the specific heparin-binding domain peptide.
(136) First we examined any background affinity the GAGs may have had for the naked streptavidin column by running the total GAG (tGAG) fraction against a column bed devoid of BMP2-HBP (
(137) Pre-incubation of tGAGs with the BMP2-HBP revealed the complete inability of the peptide to associate with the column (
(138) Column Loading Capacities
(139) As the proportion of tGAGs that were likely to have a relative affinity for the BMP2-HBP was unknown, we first sought to standardise the quantities of tGAGs loaded onto the peptide column at each run for separation. Hi-Trap columns were prepared by immobilising 1 mg of the BMP2-HBP for the extraction of tGAGs with a specific affinity for the BMP2 heparin-binding site. This amount was selected so as to maximise the quantity of available peptide for future experiments should column stability become compromised over time. Instability is a significant problem with peptide columns, with corresponding impacts on consistency. Initial attempts at loading of 25 mg of tGAGs onto a 1 mg BMP2-HBP coupled column resulted in a clear overloading, as observed via absorbance at 232 nm in the flowthrough (
(140) Further optimisation led us to routinely load no more than 6 mg of tGAGs onto a 2 mg BMP2-HBP column. This, as evidenced by the flowthrough peak (
(141) GAG Domain Analysis
(142) GAG+ Chain Specificity
(143) With the establishment of a standardised protocol, we were able to reproducibly isolate GAG+ fractions for further analysis.
(144) Given the domain structure of heparan sulfate that mediates the binding specificity for proteins, it is likely that multi-domain GAG chains that bind to the column are in fact composed of a large proportion of chain with little or no specific affinity for BMP2. Similarly, it is possible that chains that appeared GAG may in fact contain domains that carry some affinity for the BMP2-HBP. In order to examine these possibilities, it was necessary to break down the GAG chains into their component domains for more extensive examination.
(145) The enzyme heparinase III (heparitinase I) cleaves HS chains primarily in those areas flanking highly sulfated regions, thereby liberating the highly charged, protein-associating domains that bind susceptible growth factors, in this case the BMP2-HBP. Both GAG+ and GAG fractions were exposed to heparinase digestion, although neither fraction showed any change in their affinity for the BMP2-HBP (
(146) Heparinase III digestion of both full length GAG+ and GAG fractions was subsequently conducted, and both digested sample sets subsequently loaded onto the BMP2-HBP column to assess retention affinity.
(147) The efficacy of the heparinase digestion was validated by the increase in relative absorbance of samples of equal dry weight after enzymatic digestion, as shown in
(148) Interestingly, heparinase digestion of full length GAG chains yielded no fractions carrying any notable affinity for the BMP2-HBP (
(149) GAG+ Composition
(150) Full Length GAG+ Sizing
(151) In order to examine the composition of GAG+ fractions from the BMP2-HBP column, we first examined their average size. This was to ensure that we were actually separating GAG chains of reasonable length, rather than small fragments not carrying any specific affinity. Although any sizing of GAG chains is problematical, owing to their relatively rigid rod-like conformation, a set of assumptions invoking Stoke's radius and apparent sphericity can be made.
(152) Full length GAG+ samples were loaded onto Biogel P10 gel filtration columns (1 cm120 cm) with an exclusion limit of between 20 kDa to 1.5 kDa. Absorbance measured at 232 nm indicated a large proportion of GAG+ molecules had an overall apparent size greater than 20 kDa (
(153) It has been posited that sugar chains must be longer than approximately 10-14 rings in order to potentiate significant biological activity for the FGF family of mitogens. In terms of apparent molecular weight, a chain of 14 fully sulfated disaccharides corresponds to approximately 8.7 kDa. As the majority of chains found in the GAG+ samples show an apparent molecular weight >20 kDa, it is reasonable to assume that the interaction that they carry for the BMP2-HBP has some specific affinity and is not the result of a general non-specific interaction.
(154) GAG+ Sugar Species
(155) There are five major glycosaminoglycan sugar families: hyaluronan, keratan sulfate, dermatan sulfate, chondroitin sulfate and heparan sulfate. Of these five, only heparan sulfate, chondroitin sulfate and dermatan sulfate have the capacity to generate variably sulfated domains that may code for specific interactions with particular cytokines such as BMP2. The identification of the type of sugar species isolated using the BMP2-HBP column was of crucial importance for this study, and was determined using a combination of diagnostic chemical and enzymatic degradations. In particular, heparan sulfate, one of the major GAG candidates for the interaction with BMP2, can be completely degraded into its disaccharide components in the presence of nitrous acid.
(156) Thus, our HBP-retained GAG samples were incubated with nitrous acid for 20 min prior to separation on a Biogel P10 sizing column. Examination of the resulting chromatogram revealed an almost complete degradation of all GAG+ sugar samples, as measured by absorbance at 232 nm and 226 nm (
(157) This result strongly suggests that the full length sugar chains isolated specifically against the BMP2-HBP consist primarily of heparan sulfate, as other sugar chains are not affected by nitrous acid depolymerisation.
(158) Although almost all the GAG+ chains could be degraded in such a manner, a small peak was nevertheless observed at higher molecular weights (>20 kDa). It can be postulated to consist of chondroitin sulfates, of which CS-B (dermatan sulfate) and CS-E (chondroitin-4,6-sulfate) demonstrate sulfation complexity akin to heparan sulfates.
(159) GAG Species Analysis
(160) BMP2-HBP Specific GAGs (Alternative Species)
(161) The degradation of full length GAG+ chains by exposure to nitrous acid clearly indicated that the majority of GAG+ sugar chains consisted of the heparan sulfate sugar species (
(162) We tested chondroitin-4-sulfate (C4S), chondroitin-6-sulfate (C6S) and dermatan sulfate (DS) by, in each instance, loading 6 mg of the sugar onto the BMP2-HBP column under the same conditions used to isolate GAG+ chains from MC3T3 matrix samples.
(163) The chromatograms illustrating the affinity of each of the 3 sugar chain types showed that only C4S (
(164) As any potential interaction between chondroitin sulfate and BMP2 has not yet been well characterised, these results led us to question the validity of column chromatography as an accurate monitor of the BMP2/heparan interaction. In order to further explore the specificity of the interaction dynamic, we tested several commercially available sugar species for their affinity to the column. These included heparan sulfate, low molecular weight heparin (Heparin-LMW), high molecular weight heparin (Heparin-HMW) and Heparin-HMW treated with heparinase I.
(165) Interestingly, none of these commercially available GAG species appeared to demonstrate any specific interaction with the peptide column. Heparan sulfate from bovine kidney had very little affinity (
(166) None of the tested heparin samples showed even a minor affinity for the column. This is of particular interest as BMP2 itself was historically first isolated using heparin columns. In order to confirm this result, both LMW (
(167) As we surmised that the relatively small BMP2-HBP peptide may have had difficulty maintaining its association with the much larger heparin molecules, we next predigested the heparin-HMW samples using heparinase I. These smaller heparin-HMW fragments were then run over the BMP2-HBP column; this treatment did not, however, appear to improve the ability of any of the heparin samples to bind the peptide column (
(168) This inability of the peptide column to show any specific interaction with any of the various preparations of heparin was somewhat unexpected, due to BMP2 conventionally being isolated via heparin affinity. It is possible, however, that this may be as a result of the reversing of the receptor-ligand order of interaction; in this case the BMP2-HBP represented the fixed receptor as opposed to the heparin that represented the ligand, or that the concentrations of BMP2-HBP or soluble heparin favour a dissociated state that rapidly negates any affinity under flow/salt stress.
(169) Conclusions
(170) The use of a preosteoblast-derived ECM substrate provided us with a useful model for simulating the activity of natively secreted, ECM-associated GAGs in relation to such osteoinduction. Though numerous previous studies have examined the role that this native interaction has in modulating the activity of BMP2, this has usually been conducted at the level of the cytokine, rather than with a view to exploring the sequence specificity of the biomodulating GAGs.
(171) Hence here we sought to exploit the availability of natively secreted GAGs in the MX substrate and their potential for direct, sequence-specific interaction and modulation of BMP2-induced C2C12 myoblast commitment to the osteogenic lineage.
(172) Anion Exchange
(173) The use of this particular standard and well characterised protocol provided us with conclusive evidence for GAG accessibility from the NH.sub.4OH-treated MX substrate. Our initial concerns were centred around the harsh chemical treatment used to lyse the cellular components of the ECM, and that this may have also resulted in the stripping of the majority of GAGs from the ECM. However, the significant, high affinity peak observed in the anion exchange chromatogram clearly illustrates the retention of a large quantity of GAGs within the MX substrate. While this particular methodology does not allow for the identification of individual GAG species, it does offer conclusive evidence of their presence in the sample due to their being amongst the most negatively-charged molecules secreted by cells.
(174) BMP2-HBP Column System
(175) Previous research into the functional role of the BMP2 heparin-binding peptide provided us with a useful tool to investigate the potentially specific interaction that BMP2 has with GAGs. This single string of amino acids, located at the N-terminus of each BMP2 monomer, appears to be solely responsible for mediating BMP2's affinity for GAGs.
(176) We thus investigated the use of this region of the BMP2 molecule as a ligand bait in attempts to retain those GAG chains that carried relative affinity for the cytokine. The use of the BMP2-HBP in this manner resulted in a significant retention of HS to the peptide column (GAG+).
(177) Column Preparation
(178) Using an N-terminal biotinylated HBP we prepared a BMP2-HBP affinity chromatography column, and were able to successfully retain GAG samples that were candidates for controlling the native BMP2 homodimer. Initial preparations of the column highlighted some interesting problems. Preparations of biotinylated BMP2-HBP that were premixed with tGAGs showed an inability to bind to the column. As later tests showed that the BMP2-HBP easily attached to the streptavidin columm when loaded on its own this result indicated that the GAGs interfered with the ability of the peptide's biotinylation site to associate with the streptavidin column. The tGAGs themselves carried no affinity for the streptavidin, indicating that the direct interaction with the BMP2-HBP, possibly via steric hindrance, was responsible for this.
(179) Column Optimisation
(180) Without any direct information that would allow us to estimate the binding capacities of GAG+ sugars in our samples, our peptide column needed to be optimised to ensure that excessive sample loading would not lead to column saturation and consequent sample loss. This initially involved intentionally saturating the column in order to examine the binding capacity of a known quantity of BMP2-HBP. Even with a large quantity of tGAGs the peptide was capable of retaining the majority of GAG+ sugar chains. Under these conditions as little as 1 mg of BMP2-HBP was able to completely retain all GAG+ chains within two cycles. The column thus appeared to simulate a true BMP2 growth factor column and provide an extremely efficient way of extracting GAG+ samples.
(181) The optimisation of peptide-based columns for specific GAG isolation is a complex procedure that varies greatly depending on the size and individual chemical characteristics of the protein used. Previous studies, utilising FGF-1 and 2 growth factor columns (Turnbull and Nurcombe, personal communication), also showed a significant need for continual column maintenance and short viable column life-spans. These studies demonstrate the laborious nature of working with peptide columns and the care that must be taken to correctly optimise this manner of system. Unfortunately, while other systems for the analysis of specific protein-GAG interactions exist, these generally lack the capacity to isolate sufficient quantities of GAGs for further analysis, making them inappropriate for our intended course of study.
(182) GAG Domain Analysis
(183) GAG sulfation patterns are, particularly in the case of heparan sulfate (HS), frequently concentrated into domains of high sulfation that are interspaced with regions of little sulfation. This grouping of sulfation sites into domains is what provides region-specific binding of ligands to the GAG chain, allowing a single sugar molecule to potentially bind a variety of different targets, and to stabilise the interaction between these, as is seen in the FGF system. Exceptions to this proposed model for HS-ligand interactions include the interaction between interferon gamma (IFN) and heparan sulfate. In this instance the interaction between the GAG and IFN leads to an increased potency of the cytokine. IFN that remains dissociated from local GAGs is rapidly processed into an inactive form, thereby preventing its signalling in inappropriate areas after diffusion. IFN also displays four separate heparin-binding domains, each with a different sequence, a finding not unusual for heparin-binding proteins. However, only two domains found immediately at the C-terminus of the protein have been shown to mediate INF's heparin-binding characteristics. Importantly, sequence analysis of the HS sequence with specific affinity for these two IFN heparin-binding sites revealed an interesting difference in comparison to the commonly observed model of HS-ligand interaction. In this case, the sequence of HS responsible for the binding of IFN was found to be composed of a predominantly N-acetylated region, carrying little sulfation. This region was flanked by two small N-sulfated regions. This differs significantly with the system observed in FGF, where sulfation patterns in NS domains are responsible for mediating the interaction between FGF and HS. In recent years, this type of interaction has been observed in numerous other systems, such as PDGF, IL-8 and endostatin. The discovery of this kind of interaction with HS, as observed in these cytokines, may be able to explain the bioactivity observed in hyaluronan, which carries no sulfation patterns at any point along its chain and yet has the ability to modulate the activity of such factors as NF-B.
(184) These observed interactions between ligands and GAGs, in particular that of IFN, differ significantly to the proposed, and our observed, mode of interaction between HS and BMP2. BMP2's single, N-terminal heparin-binding domain exhibits no secondary structure and appears to interact with HS solely on the basis of charge. While in-depth sequence analysis of HS that binds this peptide sequence was not conducted, its requirement to be eluted under approximately 300 mM NaCl conditions lead us to suspect the presence of a moderate degree of sulfation, thereby placing this interaction within the conventional model of sulfation patterns mediating specific interactions.
(185) GAG+ Chain Specificity
(186) The allocation of sulfation patterns into domains that give HS its ability to stabilise proteomic interactions also results in the possibility that a GAG+ sugar chain of sufficient length and complexity may carry several domains that have no direct affinity for the BMP2-HBP on their own, due to their carrying a different sulfation sequence. Conversely, it is also possible that some full-length sugar chains that were identified as having little affinity for the BMP2-HBP (GAG) may contain some cryptic domains that do carry such affinity.
(187) In recent years, numerous reports have been published that provide strong evidence for a sulfation code within these complex carbohydrate chains. While the details of this sulfation code remain difficult to elucidate, and the sequencing of long chains of sulfated carbohydrates is a complex and time consuming process, a number of possible modes of specific interaction between GAGs and ligands have been proposed. One observation in particular has led to the characterisation of numerous GAG-ligand models; the grouping of sulfation into discrete regions, or domains, along the length of many types of GAGs, such as heparan sulfate. Interestingly no template for this phenomenon has yet been observed, and it appears to be primarily a result of the temporal activity of the sulfotransferase enzymes responsible for this phase of GAG synthesis.
(188) Particularly useful tools in the study of specific GAG sequences are a number of heparin lyases that can be used to examine targeted depolymerisation of complex carbohydrate chains, thereby providing insight into their structure. One particular heparan lyase, heparinase III (heparitinase), cleaves heparin sulfate chains at sites flanking the highly sulfated domains that may occur in heparan sulfate chains. Thus, using this enzyme, it is possible to liberate these potentially active regions from the full length sugar chains and separate them, if they function as single domains, via affinity chromatography, from regions with no specific affinity for the BMP2-HBP.
(189) It is important to note that, in the case of GAG-ligand interactions, affinity by sequence does not necessarily guarantee bioactivity. The mode of activity mediated by GAGs during their association with their various ligands differs greatly depending on the system. In some instances where the sugar chain is responsible for prolonging protein-protein interaction via stabilisation of tertiary protein structures, such as is found between FGF and its receptor, and the interaction between HGF/SF and Met, multiple discrete sulfation regions may be involved in mediating the intended bioactivity of the sugar chain. In such instances the isolation of individual sulfated domains from a full length carbohydrate chain may, in fact, result in an inhibition of sugar bioactivity since though each domain-fragment still binds its intended target it is unable to mediate the intended biological effect of a combined full length carbohydrate chain. Interestingly, this particular characteristic of GAG-ligand interactions is precisely what makes this manner of approach useful for modulating BMP2 activity. The proposed model for GAG modulation of BMP2 bioactivity involves immobilization of the cytokine to GAGs in the ECM or on the cell surfaces. In this type of system the application of exogenous GAGs specific to the heparin-binding domain of BMP2 would prevent this interaction, increasing short term BMP2 mediated signalling, similar to the effect observed during the addition of soluble heparin. While there is some indication that this manner of interaction would continue to protect the cytokine from proteolytic degradation, delocalization of BMP2 from its intended region of bioactivity has the potential to negatively impact the cytokines effectiveness in the long term.
(190) Control testing of our full length GAG+ and GAG chains resulted in similar profiles to those observed during their primary separation. Analysis of GAG+ and GAG chains post treatment with heparinase III, however, gave surprising results. The digestion of GAG+ chains did not seem to generate separable fragments based on simple affinity for the BMP2-HBP. Furthermore, the digestion of full length GAG chains yielded no liberation of positive domains from the negative sugar chains. There is some possibility that the enzymatic digestion did not go to completion. However, the resulting chromatogram clearly showed a large increase in the absorbance at 232 nm when compared to the full length GAG chains. As a large proportion of the absorbance of glycosaminoglycans at 232 nm is mediated via absorbance of unsaturated bonds, such as those formed during enzymatic depolymerisation, it strongly indicates that the enzymatic digestion was, in fact, successful.
(191) The implications of this result are somewhat unusual. This data suggests that GAG chains are not only synthesised by cells to specifically interact with BMP2, but that, in the case of MC3T3 cells, these sugar chains carry a number of sequence repeats specific for aspects of BMP2 metabolism. The fact that BMP2 is an extremely potent factor may offer an explanation for this observation. The effects of BMP2 on the osteoinduction of mesenchymal progenitor cells is well documented, as is its ability to induce ectopic bone formation in cells that are even more removed from the osteogenic lineage. Given this potency, aberrant signalling of BMP2 is known to have deleterious consequences both for healing and in development. It is possible that numerous repeats of the BMP2-HBP interaction sequence on preosteoblast GAGs are designed to ensure a maximal binding, and thereby the modulation, of this cytokine's ability to induce altered cell fate. Conversely, the extremely low concentrations of BMP2 produced in vivo may also require this type of sugar chain production in order to ensure the retention of a sufficient local concentration, an observation supported by the extremely high concentrations of BMP2 required in vitro to induce the osteogenic differentiation of C2C12 myoblast cells.
(192) Of particular interest is the fact that this repetition of BMP2-binding domains is produced via a synthesis pathway for which no template or timing mechanism has yet been elucidated. The accuracy and reproducibility of sequence specific domains within a single sugar chain (as opposed to the random clustering of such domains with those against other ligands) strongly suggests that these cells do, in fact, have the ability to direct the generation of specific sugar sequences. The current understanding of HS structure implicates the progressive post-synthesis editing of the carbohydrate chain in the generation of sequence-specific regions, with observations pointing towards some manner of enzymatic template, whereby the local concentrations of particular sulfotransferases as well as other interacting molecules are used to directly control the generation of specific sugar sequences. Our current understanding of this mode of specific synthesis is largely formulated based on numerous studies including those by Lindahl et al. that investigated the high affinity interaction between antithrombin III and heparin, and those by Esko et al. involving Chinese hamster ovary (CHO) cell mutants with altered GAG synthesis pathways. These studies, while varying significantly in their approaches to GAG analysis, all point towards a highly conserved system of specific GAG synthesis, for the directed modulation of cytokine and receptor activity. Importantly, these studies also serve to explain the potential generation of such BMP2 repeats as were observed in our study.
(193) GAG+ Constitution
(194) Full Length GAG+ Size
(195) The bioactivity of individual GAGs chains for FGFs is closely related to carbohydrate chain length. A common approach to assessing GAG bioactivity is to assay ever shorter sulfated domain fragments and so determine the shortest possible sequence required to mediate the activity observed.
(196) Using this approach we first examined full length GAG+ sugar chains, and determined that they were >20 KDa in size, long enough to carry multiple domains with affinity for BMP2. Interestingly, this observation provided support for the earlier observation that GAG+ samples treated with heparinase 3 showed multiple repeats of carbohydrate chain segments with a specific affinity for BMP2, since a variably sulfated sugar chain of this size has the capacity to carry numerous sulfated domains.
(197) GAG+ Sugar Species
(198) With the majority of the five glycosaminoglycan types that constitute the glycome able to encode the observed specific interactions with BMP2, it was necessary to elucidate which of these GAG types could be involved in this specific association. Although the prime candidate for this interaction is a heparan sulfate, analogous growth factor interactions have also been identified for chondroitin and dermatan sulfates.
(199) Heparan sulfate can be totally depolymerised into its disaccharide components with nitrous acid. This particular characteristic, shared with heparin and keratan sulfate, is essential for the analysis of specific GAG populations. In the case of our analysis of the carbohydrate constituents of our GAG+ samples, degradation due to nitrous acid was diagnostic of heparan sulfate. This probability is primarily due to its heparan sulfate's higher degree of charge patterning via sulfation in comparison to either heparin or keratan sulfate. Ultimately, this charge patterning is responsible for BMP2's specific interaction with HS.
(200) Our analysis utilising the nitrous acid protocol showed a complete degradation of the GAG+ sample set indicating that the majority of sugars in the GAG+ sample set were in fact 1,3-linked and, thus, were heparan sulfate. This result supports the numerous observations in regards to the specificity of heparan sulfate cytokine interactions, particularly the interaction that BMP2 exhibits with heparin and HS.
(201) GAG Species Analysis
(202) BMP2-HBP Specific GAGs (Alternative Species)
(203) The small remnant peak that was observed after the degradation of GAG+ samples by nitrous acid supports the possibility that other sulfated GAGs carrying some specific affinity for BMP2 may be found in the GAG+ sample set. Given our current understanding of the role of sulfation in mediating the interaction beween GAGs and BMP2, chondroitins and dermatans are the most likely alternative sugars to show a specific interaction with BMP2 as these show the highest potential diversity in sulfation patterns.
(204) A methodology frequently employed for GAG analysis includes examining the role of individual sulfation positions on GAG-ligand interactions. This method of analysis gives an indication of the importance of individual sulfation positions in maintaining the interaction between the GAG chain and its specific target. Furthermore, since the different species of GAGs only have the potential to carry sulfation patterns specific to their species, this can aid in narrowing the possible glycosaminoglycan candidates that may show an affinity for a specific ligand.
(205) To this end we examined the affinity for the BMP2-HBP carried by variably sulfated CS chains, C4S and C6S, and standard DS. Interestingly, only C4S carried any significant affinity for the BMP2-HBP. This data indicates that it is likely that the 4-O-sulfation is necessary for CS to interact with the BMP2-HBP. Interestingly, dermatan sulfate showed no affinity for the BMP2-HBP. This observation is of interest since DS is the only CS species that demonstrates diversity in sulfation similar to that of HS. Furthermore, our observations indicate a possibility that the epimerisation of GlcA to IdoA in DS compromises the ability of this sugar type to bind the BMP2-HBP. Both C4S and DS are able to carry 4-O-sulfation, yet only small quantities of DS were retained on the column in comparison to C4S. Alternatively, this lack of affinity may simply be due to this particular batch of DS not carrying sufficient 4-O-sulfation to effectively mediate binding to the BMP2-HBP. Interestingly, these particular observations appear to demonstrate an interaction between BMP2 and CS carrying 4-O-sulfation. While previous studies have investigated the use of CS-BMP2 interactions in drug delivery systems, not much is known about any sequence specific interaction between individual CS species and BMP2. However, since HS chains are composed of 1,4-linked disaccharide units, the observed 4-O-sulfation responsible for CS-BMP2 interactions is not found in HS-BMP2 interactions, pointing to a sequence specific interaction not found in CS. Thus it is likely that the remnant peak observed post-nitrous acid treatment may contain small quantities of 4-O-sulfate carrying C4S or DS.
(206) Further investigation revealed that neither commercial HS nor heparin held any significant affinity for the peptide column. The HS used for this assay was purchased commercially from Sigma-Aldrich and was derived from bovine kidney. Given what is known about the tissue specificity of HS it is possible that this commercially available HS, isolated from bovine kidney sources, carried negligible carbohydrate sequences required to specifically mediate an interaction with BMP2. Similarly neither LMW nor HMW heparin showed any affinity for the peptide column. The heparin used for this analysis was also purchased from Sigma-Aldrich, and was derived from porcine intestinal mucosa.
(207) While heparin's interaction with antithrombin III has been well characterised, and notwithstanding its versatile role in the isolation of susceptible molecules, heparin's interaction with growth factors is not, in general, regarded to be specific due to its uniform sulfation. However, given that heparin is routinely used to isolate BMP2, it is somewhat surprising that neither of the heparin samples interacted with the peptide column to any significant degree.
(208) A further possibility for this lack of interaction between the peptide column and heparin is due to the difference in molecular weights between the two molecules. The small BMP2-HBP attached to the column may have difficulty in maintaining its association with the larger, heavily sulfated heparin chain. The inability of heparinase-cleaved heparin to bind the column, however, appeared to indicate that the steric effects of using full length heparin on the column were not solely responsible for disrupting the potential interaction between the sugars and the BMP2-HBP. There is no immediately apparent reason for this inability for commercial heparin to associate with the BMP2-HBP column, though it may be postulated that further spatial separation of the BMP2-HBP from its associated bead via spacer chains may help to ameliorate this problem.
(209) Summary
(210) In this study we have demonstrated the use of affinity chromatography to isolate a subset of glycosaminoglycans that carry a specific affinity for the BMP2-HBP, and have shown the potential for this procedure to yield reproducible results. During this portion of our investigation into the interaction between matrix based GAGs and BMP2, we have made several observations with regards to both the type of GAGs involved in mediating this association and their structure.
(211) Our results have implicated heparan sulfate for mediating the majority of the affinity BMP2 has for the preosteoblast ECM, an interaction which is increasingly recognised as being responsible for the modulation of BMP2 activity. Furthermore, our investigation into the likely structure of the ECM-resident GAGs isolated on the basis of their affinity for the BMP2 heparin-binding site have yielded a surprising result.
(212) Our data indicates that full length BMP2 GAG+ chains do not consist of individual domains with specific affinity for BMP2 interspersed with regions of little or no affinity for the factor. Instead, our results imply that these GAG+ chains consist of multiple BMP2-binding domain repeats. This result is surprising on several levels. Firstly, the repetition required to fulfil this observation over the full length of a >20 kDa carbohydrate chain points to the presence of some manner of synthetic template. Indeed, while previous studies have been unable to derive a template for the assembly of tissue-specific GAG chains, the very fact that such specificity exists supports the presence of a template-based system. Although no genomic template has been elucidated for this process there exists some possibility of a proteomic, perhaps enzymatic, template.
(213) Secondly, this observation provides some evidence as to the importance of the interaction between BMP2 and GAGs. Multiple repeats of the BMP2 affinity site along the length of the carbohydrate chain may be required to ensure maximal binding of BMP2 to the ECM. This particular association has been shown to significantly lengthen the factor's half life, as well as probably being responsible for maintaining a significant local concentration in order to maintain signalling. Alternatively, some studies have proposed a model whereby BMP2 is spatially inhibited from interacting with its receptors due to the interactions with ECM-based GAGs. In this particular scenario the repetition of BMP2 affinity sequences would ensure a maximal binding of the factor, thus reducing the chance of it interacting with its receptors.
(214) Our cumulative results indicated that this system for the isolation of GAGs from the ECM is viable and likely to yield GAG chains that have a specific affinity for BMP2.
(215) This study supports previous findings in regards to the interaction between GAGs and BMP2. Although the prevention of BMP2 associating with the ECM in vitro through the addition of exogenous GAG+ appears to increase BMP2 signalling and upregulates osteogenic gene expression, observations to the contrary have also reported. In these studies, in vivo examination of BMP2's modulation via the HBP showed a distinct improvement in long term osteogenesis when the association with ECM GAGs was increased. It is possible that this interaction plays a major role in maintaining local concentrations by preventing the factor from diffusing away from its sites of primary activity. In light of these studies and our own observations, we propose that BMP2's activity is both positively and negatively regulated by its association with GAGs. Negative regulation may occur precisely via the model proposed by Katagiri and colleagues, whereby the retention of BMP2 in the ECM, away from its receptors, leads to a downregulation of BMP2 signalling. However, cells that require signalling by this factor may potentially secrete various enzymes to remodel extracellular sugar chains, such as sulfatases and heparinases, in order to clip away GAGs retaining BMP2 in the ECM, thereby liberating the factor and allowing it to signal, leading to the BMP2-ECM interaction ultimately becoming one of positive maintenance of the cytokine's activity. Alternatively, negative regulation of BMP2 by cell surface GAGs, may be via the internalisation of GAG chains with their associated BMP2 molecules, as has been observed by Jiao and colleagues.
(216) These previous studies, in conjunction with our own observations, have lead us to conclude that the sequence-specific interplay between BMP2 and heparin sulfate represents an intricate control mechanism that has the capacity to both positively and negatively regulate BMP2 signaling. Physiologically this interaction is responsible for enforcing context dependent responses to this potent cytokine in respect to many facets of embryonic development, precursor commitment and wound healing.
EXAMPLE 2
Purification of BMP2 Peptide Specific HS
(217) We used a peptide having heparin-binding properties from the mature BMP-2 sequence to identify novel HS that bind to the peptide.
(218) TABLE-US-00001 MatureBMP-2aminoacidsequence: [SEQIDNO:5] QAKHKQRKRLKSSCKRHPLYVDFSDVGWNDWIVAPPGYHAFYCHGECPF PLADHLNSTNHAIVQTLVNSVNSKIPKACCVPTELSAISMLYLDENEKV VLKNYQDMVVEGCGCR Heparin-bindingpeptideaminoacidsequence: [SEQIDNO:1] QAKHKQRKRLKSSCKRHP
(219) To replicate the natural presentation of the heparin-binding site we biotinylated the peptide on it's C-terminus and kept the proline (P) to improve the flexibility/accessibility of the peptide once bound to the streptavidin column.
(220) Isolation of BMP2 Peptide Specific HS
(221) Materials used included a BMP2peptide coupled Streptavidin column, HiPrep Desalting Column (GE Healthcare), 20 mM PBS+150 mM NaCl (Low Salt Buffer), 20 mM PBS+1.5 M NaCl (High Salt Buffer), HPLC grade Water (Sigma), Biologic-Duoflow Chromatography system (Bio-Rad) and a Freeze Drier.
(222) The column was equilibrated with Low Salt buffer and 1 mg Sigma HS (H9902) was dissolved in low salt buffer and passed through the BMP2-Streptavidin column. Unbound media components were removed from the column by washing low salt buffer (20 mM PBS, pH 7.2, 150 mM NaCl) until the absorbance of the effluent at 232 nm almost return to zero. HS bound to the matrix was eluted with high salt buffer (20 mM PBS, pH 7.2, 1.5 M NaCl). Peak fractions were pooled and freeze dried for 48 hrs.
(223) HS 1 mg was applied to the column and washed with 20 mM PBS buffer containing a low (150 mM) NaCl concentration. After washing with low salt buffer, the bound HS were eluted with 20 mM PBS buffer containing a high (1.5 M) NaCl concentration. Peaks representing retained fractions (monitored at 232 nm) were collected and subjected to further desalting.
(224) After freeze drying 6 mg of positive HS (GAG+) and 1.8 mg of negative HS (GAG) were obtained.
EXAMPLE 3
Evaluation of BMP-2 Specific Heparan Sulfates
(225) C2C12 are mouse mesenchymal stem cells normally exhibiting myogenic differentiation but capable of being directed in the osteogenic lineage with supplementation of BMP-2 at passage 3. C2C12 cells at passage 3 were maintained in DMEM with 1000 g/L glucose (low glucose), 10% of FCS, 1% of P/S and without L-glutamine (maintenance media).
(226) DMEM with 1000 g/L glucose (low glucose), 5% of FCS, 1% of P/S and without L-glutamine was used as differentiation media.
(227) Effect of BMP-2 on Osteogenesis
(228) We evaluated the effects of exogenous BMP-2 on osteogenesis by measuring the levels of expression of osteogenic markers (osteocalcin, osterix, Runx2).
(229) Through assaying the effect of addition of different amounts (100 ng/ml and 300 ng/ml) of BMP-2 to the cells we observed a significant decrease at day 5 in the expression of osterix, osteocalcin and Runx2 in cells having 100 ng/ml BMP-2 compared to addition of 300 ng/ml BMP-2 (
(230) Materials and Methods
(231) C2C12 cells at passage 3 were used. Cells were kept in liquid Nitrogen at Passage 3 with 110.sup.6 cells/vial. Once cells were taken from liquid Nitrogen, we added 500 l of culture media, pipetted up and down to refreeze the cells and immediately added 15 ml of culture media.
(232) Culture media was DMEM with 1000 g/L glucose (low glucose), 10% of FCS, 1% of P/S and without L-glutamine. Treatment media was DMEM with 1000 g/L glucose (low glucose), 5% of FCS, 1% of P/S and without L-glutamine.
(233) C2C12 cells were allowed to grow to 75% confluence before harvesting (normally 2 to 3 days) in culture media.
(234) Cells were counted as follows. Media was first aspirated/discarded; 15 ml of PBS added, discard the PBS and add 3 ml of trypsin, incubate at 37 C. for 5 min to lift the cells from the flask. 9 ml of culture media added to neutralize the trypsin. GUAVA used to determine the amount of cells for subsequent cell seeding onto the experiment plates. For example, for 3 sets of 12-well plates 30,000 cells36 wells3.7 cm.sup.2=4,000,000 cells. Dilute the cells from the stock and add the desired amount of culture media for cell seeding (each well requiring 500 l of media with 30,000 cells).
(235) To prepare BMP2 stock 10 g rhBMP2 (Bone Morphogenetic Protein 2) was re-suspended in 100 l of 4 mM HCl/0.1% BSA.
(236) The following RNA extraction protocol was used. 350 l of RA1 buffer was used for cell lysis. Cells were frozen with RA1 at 80 C. for one day after which cells were thawed and the lysate filtered for 1 min at 11,000 g. The filtrate was mixed with 350 l 70% ethanol in 1.5 ml tubes and centrifuged for 30 s at 11,000 g. 350 l of MDB buffer was added and the mixture centrifuged for 1 min at 11,000 g. 95 l of Dnase reaction mixture added and mixture left at room temperature for at least 15 min. Then wash with 200 l of RA2 buffer (to deactivate the Dnase), and centrifuge for 30 s at 11,000 g. Wash with 600 l of RA3 buffer, centrifuge for 30 second at 11,000 g. Wash with 250 l of RA3 buffer, centrifuge for 2 min at 11,000 g. Elute the RNA with 60 l of Rnase-free H.sub.2O, centrifuge for 1 min at 11,000 g. Measure the concentration using Nanodrop (unit in ng/l).
(237) RT (reverse-transcription) experiments were performed as follows. The following were mixed in a PCR tube: Random Primer (0.1 l), DNTP (1 l), RNA (250/500 ng), Rnase-Free H.sub.2O (topped up to a final volume of 13 l). Incubate at 65 C. for 5 min. Incubate on ice forat least 1 min. Collect the contents and centrifuge briefly before adding: 1.sup.st Strand Buffer (4 l), DTT (1 l), RnaseOUT (1 l), SSIII Reverse (1 l). Top up to final volume of 20 l. Mix by pipetting up and down. Incubate at room temperature for 5 min. Incubate at 50 C. for 60 mins. Inactivate the reaction at 70 C. for 15 min.
(238) Reverse-transcription experiments were performed twice on separate days and the PCR products pooled together and diluted to a final concentration of 2.5 ng/l for subsequent Real-Time PCR.
(239) The Real-Time PCR was performed using a TaqMan Fast Universal PCR master Mix (2) (Applied Biosystem). PCR master Mix (10 l), ABI probe (1 l), cDNA (1 l), ddH.sub.2O (8 l). GAPDH and Beta actin were used as control genes against the experimental targets OSX (osterix), OCN (Osteocalcin) and Runx2.
(240) Effect of BMP-2 Specific HS GAG+ on Osteogenesis
(241) We evaluated the effects of the BMP-2 specific HS (GAG+) isolated in Example 2 on osteogenesis by measuring the levels of expression of osteogenic markers (osterix, Runx2, alkaline phosphatase and BspII) by quantitative polymerase chain reaction (qPCR). A time course was prepared to compare the expression of the markers over a course of 10 days to compare the control to a low and a high dose of BMP-2, the high dose being the optimal conditions to induce differentiation of the cells.
(242) Materials and methods
(243) Cells were seeded at 30,000 cell/cm.sup.2 in maintenance media and left to attach overnight. The following day we switched to differentiation media with: No additives 100 ng/ml BMP-2 (positive control) 100 ng/ml BMP-2+30 g/ml GAG (Neg GAGs) 100 ng/ml BMP-2+30 g/ml +GAG (Pos GAGs) 100 ng/ml BMP-2+30 g/ml Heparin (Sigma #H3149) 100 ng/ml BMP-2+30 g/ml Total Heparan Sulfate (Sigma #H9902HS prior to fractionation)
(244) The carbohydrates and BMP-2 were mixed together in the smallest volume possible and incubated at room temperature for 30 minutes before their addition to the media and on the cells.
(245) After 5 days, RNA was extracted using the Macherey-Nagel kits and Reverse-Transcription was performed.
(246) As we show in
EXAMPLE 4
(247) MC3T3-E1 (s14) preosteoblast cells (a mouse embryo calvaria fibroblast cell line established from the calvaria of an embryo) were expanded in MEM media supplemented with 10% FCS, 2 mM L-glutamine, 1 mM sodium pyruvate and Penicillin/Streptomycin every 72 hours until sufficient cells were generated for plating. The cells were differentiated by plating at 510.sup.4 cells/cm.sup.2 in MEM media supplemented with 10% FCS, 2 mM L-glutamine, 25 g/ml ascorbic acid, 10 mM -glycerol phosphate and Penicillin/Streptomycin. The media was changed every 72 hours for 8 days at which point the cells and media were harvested. The media was retained and clarified by high speed centrifugation and filtration through a 0.4 m filter. The cell layer was disrupted using a cell scraper and an extraction buffer containing PBS (150 mM NaCl w/o Ca.sup.2+ and Mg.sup.2+), 1% CHAPS, 8 M Urea and 0.02% NaN.sub.3.
(248) At all stages (unless otherwise stated), samples were clarified before loading onto column systems. This process included high speed centrifugation at 5000 g for 30 min, and filtration through a 0.4 m syringe filter. The samples were always clarified directly prior to loading through the column system to prevent precipitates forming in stagnant solutions.
(249) Anion exchange chromatography was used to isolate proteoglycosaminoglycan (PGAG) fractions from both the media and cell layer samples. In each case, the media or cell layer samples were run through a Pharmacia XK 26 (56-1053-34) column packed with Capto Q Anion Exchange Beads (Biorad) at a flow rate of 5 ml/min on a Biologic DuoFlow system (Biorad) using a QuadTec UV-Vis detector. The samples were loaded in a low salt buffer containing PBS (150 mM NaCl w/o Ca.sup.2+ and Mg.sup.2+), 100 mM NaCl, 0.02% NaN.sub.3 at pH 7.3. The samples were eluted in a high salt buffer containing PBS (150 mM NaCl w/o Ca.sup.2+ and Mg.sup.2+), 850 mM NaCl and 0.02% NaN.sub.3 at pH 7.3. The relevant fractions were collected and pooled into a single PGAG sample and lyophilized in preparation for desalting.
(250) The PGAG sample was desalted through four sequentially joined Pharmacia HiPrep 26/10 (17-5087-01) columns at a flow rate of 10 ml/min on a Biologic DuoFlow system (Biorad) using a QuadTec UV-Vis detector. The relevant fractions were collected and pooled into a single sample set and lyophilized in preparation for further treatment.
(251) In the fourth step, the PGAG sample set obtained from the desalting procedure was subjected to a pronase and neuraminidase treatment, in order to digest away core proteins and to subsequently liberate GAG chains. In this respect, lyophilized PGAG samples were resuspended in a minimum volume of 25 mM sodium acetate (pH 5.0) and clarified by filtration through a 0.4 m syringe filter. The total sample volume was dispensed into 10 ml glass tubes in 500 l aliquots. To this aliquot was added 500 l of 1 mg/ml neuraminidase before the mixture was incubated for 4 hours at 37 C. Following incubation, 5 ml of 100 mM Tris-acetate (pH 8.0) was added to each sample. An additional 1.2 ml of 10 mg/ml pronase, reconstituted in 500 mM Tris-acetate and 50 mM calcium acetate (pH 8.0), was added to each sample before the mixture was incubated for 24 hrs at 36 C. Following this treatment, all volumes were combined and prepared for anion exchange chromatography by centrifugation and filtration.
(252) In a fifth step, the GAG sample isolated following protein cleavage was eluted through a Pharmacia XK 26 (56-1053-34) column packed with Capto Q Anion Exchange Beads (Biorad) at a flow rate of 5 ml/min on a Biologic DuoFlow system (Biorad) using a QuadTec UV-Vis detector. In this respect, the sample was loaded in a low salt buffer containing PBS (150 mM NaCl w/o Ca.sup.2+ and Mg.sup.2+) and 0.02% NaN.sub.3 at pH 7.3. The sample was eluted in a high salt buffer containing PBS (150 mM NaCl w/o Ca.sup.2+ and Mg.sup.2+), 850 mM NaCl and 0.02% NaN.sub.3 at pH 7.3. The relevant fractions were pooled, lyophilized and desalted as per the aforementioned protocol for desalting the PGAG sample.
(253) N-terminal biotinylated peptide (1 mg), corresponding to the heparin-binding domain of BMP-2, and comprising an amino acid sequence represented by QAKHKQRKRLKSSCKRH [SEQ ID NO:6], was mixed with low salt buffer containing PBS (150 mM NaCl w/o Ca.sup.2+ and Mg.sup.2+). The mixture was eluted through a column packed with a streptavidin-coated resin matrix. The column was then exposed to a high salt buffer containing PBS (150 mM NaCl w/o Ca.sup.2+ and Mg.sup.2+), 850 mM NaCl and 0.02% NaN.sub.3 at pH 7.3, to ascertain whether, under those conditions the peptide had bound securely to the matrix. No substantial loss of peptide from the column was observed. The column was subsequently washed with the low salt buffer in preparation for sample loading.
(254) The GAG mixture (2 mg), isolated using the procedure outlined in Example 1, was suspended in low salt sodium phosphate buffer (1 mL), and loaded onto the peptide column of Example 2. The sample was eluted with a low salt buffer containing PBS (150 mM NaCl w/o Ca.sup.2+ and Mg.sup.2+). A peak corresponding to GAGs with negligible BMP-2 affinity was observed in the UV-Vis detector trace. The column fractions responsible for giving rise to this peak were combined. These fractions are known as GAGthe minus sign denoting the lack of affinity with the column. When it became evident from the UV-Vis detector that the trace had flattened to the baseline, and that no further oligosaccharide was eluting, the eluting solvent was changed to a high salt buffer containing PBS (150 mM NaCl w/o Ca.sup.2+ and Mg.sup.2+), 850 mM NaCl and 0.02% NaN.sub.3 at pH 7.3. Following this change in the eluting solvent, a peak corresponding to BMP-2 specific GAGs was observed in the UV-Vis detector trace. The column fractions responsible for giving rise to this peak were combined. These fractions are known as GAG+the plus sign denoting the presence of affinity with the column. In the case of GAG compounds sourced from preosteoblast cells, the GAG+ fraction represented 10% of the overall GAG mixture.
EXAMPLE 5
(255) The addition of BMP2 has a clearly defined capacity to induce osteogenic differentiation in C2C12 myoblasts. Similarly, the pre-incubation of BMP2 with heparin has been shown to both extend the cytokines half life and its immediate potency in vitro. Here we examined the capacity of GAG+ and GAG fractions to augment the osteoinduction of C2C12 cells in vitro by BMP2.
(256) The GAG+ sample from Example 4 (0, 10, 100, 1000 ng/mL) was added to C2Cl2 myoblasts in vitro in the presence of BMP-2 (0, 50, 100 ng/mL). Measurement of the relative expression of the osteocalcin gene indicated that the GAG+ sample was able to potentiate BMP-2 to effect osteocalcin gene expression at levels of BMP-2 far below those currently used in therapy (300 ng/mL). The results of this assay (including calculated p-values and errors) are represented graphically in
(257) Interestingly, while 1000 ng/ml of GAG+ is able to significantly augment BMP2 mediated osteocalcin expression, the addition of concentrations of GAG+ below 1000 ng/ml appear to progressively inhibit this expression. Furthermore, the addition of sufficient GAG+ also managed to drive the induction of osteocalcin by 50 ng/ml of BMP2 above that of 100 ng/ml of BMP2 on its own, indicating the potency of this interaction.
(258) This cell culture based analysis demonstrated that the addition of GAG+ to C2C12 osteogenic cultures together with BMP2 resulted in a significant upregulation of osteocalcin expression indicating an increase in BMP2 signalling efficacy. This result supports the specific association of GAG+ chains with BMP2, thereby blocking the BMP2-HBP and preventing its association with matrix-based PGAGs. The resulting upregulation of osteogenic gene expression is comparable to that observed in previous studies utilising heparin to achieve a similar effect. Interestingly, the addition of concentrations of GAG+ that fall below 1000 ng/ml appear to have an initially antagonistic effect on BMP2 signalling.
(259) One possible hypothesis to explain this observation revolves around the capacity for a given number of GAG+ molecules to bind a certain number of BMP2 molecules. Under conditions where no exogenous GAG+ is added to the culture system the majority of BMP2 molecules will be able to associate with the ECM, thereby being localised away from their cognate receptors and being unable to immediately initiate signalling. Subsequent dissociation of BMP2 from the ECM, both spontaneously and by targeted enzymatic alteration of their associated GAG chains, has the capacity to induce long term BMP2 signalling. The addition of a large number of GAG+ molecules to this system, as is the case in samples supplemented with 1000 ng/ml of GAG+, permits the majority of BMP2 molecules to remain in solution where they are free to mediate receptor dimerisation and induce downstream signalling. Both these processes of cytokine/receptor interaction likely require particular concentration thresholds in order maintain an efficient level of signalling. Under culture conditions containing 50 ng/ml of BMP2, the addition of low concentrations of GAG+ allows for a portion of the available cytokine to remain soluble while the remaining portion associates with the ECM. Under these conditions only a small quantity of BMP2 remains soluble but, due to its low concentration, becomes highly diffuse in the media leading to negligible signalling. Similarly, due to a portion of the BMP2 remaining solubilised, a reduced quantity of BMP2 can be found in the ECM, resulting in a decrease in signalling from BMP2 liberated from the ECM by direct cellular activity. However, under culture conditions containing 100 ng/ml of BMP2 the combined effects of soluble and ECM based BMP2 are, with the addition of 100 ng/ml of GAG+, sufficient to induce BMP2 signalling similar to control levels. Without further study, however, the dynamics involved in BMP2/GAG+ signalling remain unclear. Future studies utilising surface plasmon resonance may help elucidate the efficiency of BMP2/GAG+ interactions and may aid in clarifying these observations.
EXAMPLE 6
(260) The enzyme heparanase 3 was used to cleave GAG+ and GAG sugar chains from Example 4 according to the following method. GAG+ and GAG were each treated separately at a concentration of 4 mg/mL, with heparanase 3 (250 mU enzyme per 100 g oligosaccharide) for 16 hours at 37 C. Subsequently, the mixture was heated for 5 minutes at 70 C. to inactivate the heparanase 3. The digested GAG+ and GAG mixtures were each subjected to the peptide column separately. The UV-Vis detector trace of each chromatographic run indicated that the digested material showed the same affinity for the column as the undigested material.
EXAMPLE 7
(261) Coupling of Biotinylated Peptide to Streptavidin Column
(262) Method: BMP2 HBpeptide was dissolved in 20 mM phosphate buffer, 150 mM NaCl (Low Salt Buffer), at a concentration of 1 mg/ml. The peptide solution was subjected to affinity chromatography on a streptavidin column (1 ml) equilibrated in low salt buffer using a low-pressure liquid chromatography (Biologic-Duoflow chromatography system from Bio-Rad). The medium was loaded at a flow rate of 0.2 ml/min and the column washed with the same buffer until the baseline reached zero. To check that the peptide actually attached to the column, the column was eluted with a step gradient of 1.5 M NaCl (high salt buffer) and re-equilibrated with low salt buffer.
(263) The BMP2 heparin binding site (5 mg) sequence (QAKHKQRKRLKSSCKRHP-NHET biotin (SEQ ID NO: 1)) was synthesized and coupled to the 1 ml streptavidin column (GE Healthcare). The chromatogram (
(264) Purification of BMP2Specific Heparan Sulfate
(265) Method: Celsus HS was dissolved in 20 mM phosphate buffer, 150 mM NaCl (Low Salt Buffer), at a concentration of 1 mg/ml. The peptide solution was subjected to affinity chromatography on a streptavidin column (1 ml) equilibrated in low salt buffer using a low-pressure liquid chromatography (Biologic-Duoflow chromatography system from Bio-Rad). The medium was loaded at a flow rate of 0.2 ml/min and the column washed with the same buffer until the baseline reached zero. The bound BMP2 specific HS was eluted with a step gradient of 1.5 M NaCl (high salt buffer), the peak factions were collected, and the column re-equilibrated with low salt buffer. The elution peak (BMP2+ve) and flow through peak (BMP2ve) HS were collected separately, freeze-dried and stored at 20 C.
(266) The chromatogram (
(267) Desalting of BMP2 Peptide Column Bound HS
(268) Method: BMP2 specific HS was dissolved in 10 ml distilled water. The samples were subjected to desalting chromatography on a Hi-prep desalting column (10 ml) equilibrated in distilled water using a low-pressure liquid chromatography (Biologic-Duoflow chromatography system from Bio-Rad). The HS was loaded at a flow rate of 10 ml/min and the column washed with distilled water. The pure HS fractions were collected, freeze-dried and stored at 20 C.
(269) The chromatogram (
(270) Desalting of BMP2 Peptide Column Unbound HS
(271) Method: The non-specific HS was dissolved in 10 ml distilled water. The samples were subjected to desalting chromatography on a Hi-prep desalting column (10 ml) equilibrated in distilled water using a low-pressure liquid chromatography (Biologic-Duoflow chromatography system from Bio-Rad). The HS was loaded at a flow rate of 10 ml/min and the column washed with distilled water. The pure HS fractions were collected, freeze-dried and stored at 20 C.
(272) The chromatogram (
(273) SAX-HPLC Disaccharide AnalysisBMP2 Positive HS
(274) Method: Samples (100 g) were dissolved in 100 mM sodium acetate/0.2 M calcium acetate, pH 7.0. Heparinase, heparitinase I and II were all used at a concentration of 10 mU/ml in the same buffer. Each sample was sequentially digested for a recovery of disaccharides for SAX-HPLC analysis; for this the samples were digested at 37 C. as follows: heparinase for 2 h, heparitinase I for 1 h, heparitinase II for 18 h, and finally an aliquot of each lyases for 6 h. Samples were run on a BioGel P-2 column (1120 cm) equilibrated with 0.25 M NH.sub.4HCO.sub.3. The disaccharide peak was lyophilized and then dissolved in acidified water (pH 3.5 with HCl). This was passed over a ProPac PA-1 SAX-HPLC column (Dionex, USA), attached to a high pressure liquid chromatography system and the HS disaccharides eluted with a linear gradient 0 to 1.0 M NaCl, pH 3.5, over 60 min at a flow-rate of 1 ml/min. The peaks identified using HS disaccharides standards (Seikagaku, Tokyo, Japan and Iduron) monitored at A.sub.232 nm.
(275) The HS retained by the BMP2 peptide affinity column was subjected to an enzymatic disaccharide analysis by exposing it to a combination of heparin lyases (heparinase, heparitinase I and II) to completion and then subjecting the resulting disaccharide fragments to strong anion exchange HPLC (SAX-HPLC). The peaks on the chromatogram (
(276) SAX-HPLC Disaccharide AnalysisBMP2 Negative HS
(277) The HS that did not the BMP2 peptide affinity column was subjected to an enzymatic disaccharide analysis by exposing it to a combination of heparin lyases (heparinase, heparitinase I and II) to completion and then subjecting the resulting disaccharide fragments to strong anion exchange HPLC (SAX-HPLC). The peaks on the chromatogram (
(278) SAX-HPLC Disaccharide Profile of Celsus Total HS
(279) The Total HS bought from Celsus as the starting material was also subjected to a enzymatic disaccharide analysis by exposing it to a combination of heparin lyases (heparinase, heparitinase land II) to completion and then subjecting the resulting disaccharide fragments to strong anion exchange HPLC (SAX-HPLC). The peaks on the chromatogram (
(280) ESPRAnalysis of BMP2 Positive and BMP2 Negative HS
(281) Method: BMP2 +ve and ve HS (10 mg/ml) was dissolved in 1 ml 0.1 M MES, pH 5.5 and 300 l 0.1 MES, pH 5.5, containing 2 mg/ml biotin-LC-hydrazide (Pierce), EDC (7 mg) was added to the mixture and incubated at room temperature for 2 h before addition of another 7 mg of EDC. After a further 2 h incubation, unincorporated biotin was removed with a desalting column (Amersham Pharmacia). The BMP2 +ve and ve HS were tested for their capacity to bind soluble BMP2. Real time binding analysis was carried out using SPR, wherein biotin thiol-coated gold sensor chips were used as a platform for immobilized streptavidin. Using a biotin-streptavidin-biotin bridge, biotinylated HS could be immobilized on the sensor chip. The growth factor (200 nM) was then added to the solution bathing the immobilized HS and incubated for 20 min. Real time binding) was monitored by measuring the change in the minimum reflectance angle () over time.
(282)
(283) BMP2 Binding Capacity of BMP2 +ve and BMP2 ve Celsus HS Preparations Coated on an Iduron Heparin/GAG Binding Plate
(284) Method: BMP2 was dissolved in Blocking Solution (0.2% gelatin in SAB) at a concentration of 3 g/ml and a dilution series from 0-3 g/ml in Blocking Solution established. Dispensing of 200 l of each dilution of BMP2 into triplicate wells of Heparin/GAG Binding Plates pre-coated with heparin; incubated for 2 hrs at 37 C., washed carefully three times with SAB and 200 l of 250 ng/ml biotinylated anti-BMP2 added in Blocking Solution. Incubation for one hour at 37 C., wash carefully three times with SAB, 200 l of 220 ng/ml ExtrAvidin-AP added in Blocking Solution, Incubation for 30 mins at 37 C., careful washing three times with SAB and tap to remove residual liquid, 200 l of Development Reagent (SigmaFAST p-Nitrophenyl phosphate) added. Incubation at room temperature for 40 minutes with reading at 405 nm within one hour.
(285) The specially-prepared plate surface (Iduron) adsorbs GAGs without modification whilst retaining their protein binding characteristics. Binding occurs at room temperature from physiological buffers. The results (
(286) ALP Activity of BMP2 Positive and Negative HS on C2C12 Cells
(287) Methods: ALP Assay. C2C12 cells were plated at 20,000 cells/cm.sup.2 in a 24-well plate in DMEM (Sigma-Aldrich Inc., St. Louis, Mo.) containing 10% FCS (Lonza Group Ltd., Switzerland) and antibiotics (1% Penicillin and 1% Streptomycin) (Sigma-Aldrich Inc., St. Louis, Mo.) at 37 C./5% CO.sub.2. After 24 hours, the culture media was switched to 5% FCS low serum media containing different combinations of 100 ng/mL BMP2 (R&D Systems, Minneapolis, Minn.), 3 mg/mL Celsus HS and varying concentrations of BMP2-specific (+ve HS) and non-specific (ve HS) Celsus HS preparations. Cell lysis was carried out after 3 days using RIPA buffer containing 1% Triton X-100, 150 mM NaCl, 10 mM Tris pH 7.4, 2 mM EDTA, 0.5% Igepal (NP40), 0.1% Sodium dodecyl sulphate (SDS) and 1% Protease Inhibitor Cocktail Set III (Calbiochem,Germany). The protein content of the cell lysate was determined by using BCA protein assay kit (Pierce Chemical Co., Rockford, Ill.). ALP activity in the cell lysates was then determined by incubating the cell lysates with p-nitrophenyiphosphate substrate (Invitrogen, Carlsbad, Calif.). The reading was normalized to total protein amount and presented as relative amount to the group containing BMP2 treatment alone.
(288)
(289) ALP Staining
(290) Method: ALP Staining. C2C12 cells were cultured as described above. After 3 days of treatment, the cell layer was washed in PBS and stained using Leukocyte Alkaline Phosphatase Kit (Sigma-Aldrich Inc., St. Louis, Mo.) according to manufacturer's specification. Briefly, the cell layer was fixed in citrate buffered 60% acetone and stained in alkaline-dye mixture containing Naphthol AS-MX Phosphatase Alkaline and diazonium salt. Nuclear staining was performed using Mayer's Hematoxylin solution.
(291) BMP-2 specific HS (+ve HS) enhanced alkaline phosphatase (ALP) activity induced by BMP-2 at a greater degree compared to non-specific HS (ve) when evaluated histochemically (
(292) BMP2 Stability
(293) Method. Smad 1/5/8 Phosphorylation. C2C12 cells were plated at 20,000 cells/cm.sup.2 in a 24-well plate in DMEM (Sigma-Aldrich Inc., St. Louis, Mo.) containing 10% FCS (Lonza Group Ltd., Switzerland) and antibiotics (1% Penicillin and 1% Streptomycin) (Sigma-Aldrich Inc., St. Louis, Mo.) at 37 C./5% CO2. After 24 hours, the culture media was switched to 5% FCS low serum media. Treatment conditions containing 100 ng/mL BMP2 (R&D Systems, Minneapolis, Minn.) in the presence/absence of 3 mg/mL of heparin (Sigma-Aldrich Inc., St. Louis, Mo.) or BMP2-specific (+ve HS) Celsus HS were added 24 hours after the cells have been equilibrated in low serum media. Cell lysate was harvested in 1 Laemmli buffer at 0, 24, 48 and 72 hour time points. The lysate was separated in NuPAGE Novex 4-12% Bis-Tris Gel (Invitrogen, Carlsbad, Calif.) and analyzed with western blot using antibodies against Phospho-Smad 1/5/8 (Cell Signaling, Danvers, Mass.) and Smad 1/5/8 (Santa Cruz Biotechnology Inc., Santa Cruz, Calif.).
(294) The ability of the BMP2-binding HS, i.e. HS3, to prolong the effects of BMP2 on cells (presumably in part by protecting the protein against proteolytic degradation) was compared to the effects of commercial heparin. C2C12 cells were exposed to nothing, BMP2 alone, BMP2+Heparin or BMP2+HS3 for 72 hours and the levels of phosphorylation of the BMP2-specific intracellular signaling molecule Smad1/5/8 monitored by immunoblotting (
EXAMPLE 8
(295) This experiment was designed to investigate whether HS3, when combined with Smith & Nephew's bone void filler JAX gel+Tri-calcium phosphate (TCP) stars can speed up long bone repair.
(296) A non union critical defect is created in the ulna of adult rabbits, the stars placed in the defect, the wound closed and repair monitored after 4 and 8 weeks with a combination of histology and imaging.
(297) Biomaterials
(298) JAX is a -tricalcium phosphate (TCP) synthetic bone substitute manufactured by Smith and Nephew Orthopaedics Ltd, USA. JAX consists of six-armed granules, which interlock to provide 55% intergranular porosity in a defect site, allowing cell and vascular infiltration. The clinical indication is for non-load bearing bony defects of 4-5 cm. JAX also includes a hydrogel component.
(299) In Vitro Study
(300) The release of heparin at either high or low Concentrations (as a substitute for HS) from Jax gel/TCP mixtures in vitro was assessed by first labeling it with Alexa Fluor 488 dye and then monitoring its release into PBS in culture plates. Release in both cases was rapid and bursting.
(301) Fluorescent labeling of heparin. For non-biological in vitro assays, heparin, a hypersulfated member of the HS glycosaminoglycan family, was conjugated with Alexa Fluor 488 (A488, Molecular Probes, UK) using a method published previously by our group (E. V. Luong, L. Grondahl, V. Nurcombe, S. Cool. In vitro biocompatibility and bioactivity of microencapsulated heparan sulfate Biomaterials 2007; 28:2127 2136). Briefly, 3 mg of heparin (H-3149) was solubilized in 300 L of 0.1 M solution of 4-morpholinoethanesulfonic acid (MES, M3671) buffer (pH 4.5) and combined with 50 L of a 10% 1-ethyl-3-(3-dimethylaminopropryl)carbodiimide hydrochloride (EDC, Fluka 03449) solution in 0.1 M MES buffer. Subsequently, a 1% A488 solution (50 l) in 0.1 M MES buffer was added to the heparin/EDC solution. The mixture was protected from light and incubated overnight at room temperature. The fluorescently conjugated heparin was eluted on an Amersham PD10 desalting column. The labeling efficiency was approximately 1.3 mol A488/mol heparin.
(302) Release profile. Three JAX granules were loaded with either 17 or 170 g of A488-heparin (50 L in 100 L hydrogel), protected from light, and placed in 1 mL of PBS at 37 C. for 48 h. At 1, 2, 3, 4, 5, 6, 24 and 48 h, 100 L of conditioned phosphate buffered saline (PBS) was collected for sampling and replaced with fresh PBS. The concentration of released A488-heparin was quantified by fluorometry and cumulative release of A488-heparin was reported as a percentage of loading concentration (
(303) In Vivo Study
(304) Experimental Design. (See
(305) X-Ray Monitoring of New Bone Formation in Defect Sites
(306) HS3 was applied in Jax gel at two different concentrations (30 and 100 ugcalled HS30 and HS100) and assessed for new bone formation compared to no treatment over 0, 4 and 8 weeks. The HS-treated animals show clear indication of new bone formation over the controls.
(307) Radiographic Analyses. JAX granules are radio-opaque and therefore difficult to distinguish from new bone in the defect site on 2D x-rays. However, at the early time points, voids between the granules and immediately adjacent to the radius are clearly visible and the progression of bone formation in these spaces can be monitored (S. A. Clarke, N. L. Hoskins, G. R. Jordan, D. R. Marsh. Healing of an ulnar defect using a proprietary TCP bone graft substitute, JAX, in association with autologous osteogenic cells and growth factors. Bone 2007; 40: 939-947). An Imaging Radiographic System (MUX-100, Shimadzu) was used to capture 2D images of the ulna defects immediately after the surgery and at weeks 4 and 8. Digital micrographs are then taken of the X-rays. X-rays were taken under general anesthesia. X-ray micrographs are shown in
(308) Micro CT Monitoring of New Bone Formation in Defect Sites
(309) HS3 given at doses of 30 and 100 ug (HS30 and HS100) at the time of surgery was compared to PBS star alone controls using micro CT (computerized tomography) imaging (
(310) Micro-CT analyses. At weeks 4 and 8, harvested ulnas were scanned with a micro CT scanner (Skyscan 1076; Skyscan, Belgium). Scanning was performed with a resolution of 35 m and a scanning width of 68 mm. The scanner was set at a voltage of 104 kV and a current of 98 A. Cone-Beam CT-reconstruction A Sasov software (Skyscan) was used to convert the isotropic slice data obtained into 2D images. For this reconstruction, the lower and upper threshold values for bone were assumed to be 315 and 543 Hounsfield units. The data was then analyzed and remodeled using the associated CTAn software (Skyscan) for quantification and Mimics 11.1 software (Materialise, Belgium) to render 3D images. A cylindrical region of interest (ROI, cocentrically positioned over the defect site) and the total number of slices (corresponding to the length of the defect) was kept constant for all the samples. The total volume of newly formed bone within the ROI was measured by assigning predetermined thresholds for total bone content, cortical bone (JAX and radii) and trabecular bone (or newly formed bone). The data was reported as bone volume/total volume (%).
(311) HS3 (at both 30 g and 100 g doses) significantly increased the BV/TV (%) as compared to controls. There was no significant difference between HS30- and HS100-treated ulnas (
(312) Histology
(313) After the designated experimental periods, bone was harvested, fixed, decalcified, sectioned and mounted for staining with various dyes.
(314)
(315) Higher magnification H&E-stained micrographs (
(316) Histological analyses. The extracted ulnas were fixed in 10% neutral buffered formalin for 1 week under vacuum, and decalcified in 15% EDTA, pH 7.2, for 4 weeks at room temperature. Then, the ulnas were processed using a vacuum infiltration processor (Sakura Finetek, Japan) with a 14 h program. Afterdehydration and clearing, the bones were embedded in Paraplast paraffin wax (Thermo Scientific) and the paraffin blocks sectioned longitudinally at 5 M using a rotary microtome (Leica Microsystems, Germany). Paraffin sections were placed on positively charge microscope slides, dried, stained with Hematoxylin/Eosin and Modified Tetrachrome and finally examined under an Olympus Stereo (SZX12) and upright fluorescence microscope (BX51).
(317)
(318) Higher magnification Ralis Tetrachrome-stained micrographs (
(319) Immunostaining
(320) After the designated experimental periods, bone was harvested, fixed, decalcified, sectioned and mounted for staining with various dyes.
(321) Higher magnification (
(322) Immunohistochemistry analysis. Deparaffinised sections were washed with PBS, incubated with Protease XXIV (BioGenex, San Ramon, USA) for 10 min for antigen retrieval, followed by incubation with 0.3% hydrogen peroxide in water for 20 min at room temperature. After washing, sections were blocked with 5% normal goat serum in PBS for 30 min. Tissue sections were incubated with appropriate concentrations of primary antibodies: osteocalcin (ab13420, 1:150, Abcam, UK) or the same concentration of mouse IgG (MG100, Caltage Lab, USA; as negative controls) in blocking buffer overnight at 4 C. Sections were washed three times with PBS, and then incubated with rat absorbed biotin-labeled anti-mouse IgG (Vector Lab Inc, USA) for 1 h. Sections were washed with PBS and incubated with avidin-biotinperoxidase complex (ABC) solution (Immunopure ABC preoxidase staining kit, Vector Lab. Inc) for 1 h. Peroxidase activity was detected using 3,3-diaminobenzidiine-tetrahydrochloride (DAB; DAKO, USA). Sections were washed, mounted and examined under bright field microscopy using an Olympus SZX12 stereomicroscope.
(323) Torsional Testing
(324) After the designated experimental periods, bones were tested for their mechanical strength.
(325) Torsional Testing. After sacrifice at 8 weeks post-surgery, the rabbit ulnas were retrieved, wrapped in PBS-soaked gauze to maintain moisture, and frozen at 20 C. until torsional testing. Upon thawing, the ulna ends were potted in polymethylmethacrylate (Meliodent Rapid Repair, Heraeus Kulzer), contained within customized plastic blocks and allowed to solidify, to enable stable fixation. Ulnas were subsequently mounted in a MTS 858 Mini Bionix II testing system (MTS, Eden Prairie, Minn.). Polymer blocks and gauze were gently removed prior to testing. Each specimen was then tested to failure in torsion and the resulting torqueangular displacement curves were recorded. The rotation rate used was 1 degree per second until 35 degrees was reached and data were collected at 100 Hz. The stiffness, maximum torque, and angle at failure were recorded for each specimen, with the stiffness being measured as the slope of the linear portion of the torqueangular displacement curve (M. Bostrom, J. M. Lane, E. Tomin, M. Browne, W. Berberian, T. Turek, J. Smith, J. Wozney, T. Schildhauer. Use of bone morphogenetic protein-2 in the rabbit ulnar non-union model. Clin Orthop Relat Res 1996; 327: 272-282).
(326) Statistical Analyses. Quantitative data was obtained in triplicates and reported as meansstandard deviation. Statistical analyses were performed using the Student's t-test (GraphPad software), and a p-value of less than 0.05 was considered significant.
(327) Quantification of stiffness and maximum torque assessed from control and HS-treated ulnae. Stiffness was markedly improved for the HS-treated bones. As the stars occupy the largest proportion of the defect, which became a physical barrier for new bone infiltration, it resulted in improvements that were only marked (as opposed to significant)see
EXAMPLE 9
Evaluating Bone Regeneration in a Critical Sized Defect Induced by HS3-Loaded Collagen Sponges
(328) The same overall approach to that of Example 8 was used in a second study, except that the Jax TCP stars were replaced with FDA-approved collagen sponges.
(329) Biomaterials. Collagen sponges were purchased from Integra Life Sciences (HELISTAT, Integra Life Sciences Corp, USA) and measured 7215 mm. These sponges were processed from bovine deep flexor tendon, are bioabsorbable and non-pyrogenic.
(330) The morphology of the sponges was evaluated using Scanning Electron Microscopy (SEM). Briefly, collagen sponges were sputtered-coated with gold and then examined using SEM (Jeol JSM 5310 LV) at an accelerating voltage of 10 kV.
(331) Biomolecules. Heparan sulfates (HS) tested in this study were bone-morphogenetic protein (BMP) specific HS, also known as HS3.
(332) In Vivo Study
(333) The study used HS3 loaded at 30 g per sponge (HS30) and BMP-2 loaded at 10 g per sponge (BMP2-10). Bilateral ulna defects were created and treated with HS30, BMP2-10, or HS30=BMP2-10, or PBS controls.
(334) HS3 (30 g) was applied in collagen sponges either alone or in combination with BMP2 (10 g) and assessed for new bone formation compared to no treatment over 0, 4 and 8 weeks. These were assessed against the negative collagen sponge control, and the positive BMP2 control.
(335) Experimental Design. Twenty male New Zealand White rabbits (weighing 2-2.5 kg) received bilateral ulna defects. Each defect was randomly assigned to one of three experimental groups. Every defect received 1 collagen sponge soaked with one of the following treatments (total 300 L, in PBS): 30 g HS, 10 g BMP-2 (sometimes called BMP10), 30 g HS+10 g BMP-2 or an equal volume of PBS. After 4 and 8 weeks of implantation, the rabbits were sacrificed and ulnas were harvested. Four ulnas per treatment were assessed non-destructively using 2D X-rays and micro-computed tomography (micro-CT) for mineral formation at both time-points. Subsequently, these ulnas were processed for histology and immunohistochemistry. At week 8, an additional three samples per treatment were included for evaluation by torsional testing. Some defects were left empty to serve as internal controls to ensure that the model was truly non-union.
(336) Surgical Procedures. The research protocol for performing bilateral ulna osteotomies in rabbits was approved by the Institutional Animal Care and Use Committee, following all appropriate guidelines. All surgical procedures were carried out under general anesthesia and aseptic conditions. Anesthesia consisted of a combination of ketamine (75 mg/kg) and xylazine (10 mg/kg) injections as well as isoflurane via an induction chamber and facemask for maintenance. A 6 cm skin incision was made and the overlying muscle layers were parted until the length of the ulna was exposed. A 1.5 cm longitudinal defect in the central diaphysis was created using an Acculan.
(337) Radiographic Analyses. An Imaging Radiographic System (MUX-100, Shimadzu, Japan) was used to capture 2D images of the ulna defects immediately after the surgery and at weeks 4 and 8, Digital micrographs (
(338) X-ray monitoring after 4 weeks reveals significant new bone filling the defect sites in all the treatment cases as compared to the negative controls (
(339) X-ray monitoring after 8 weeks reveals the achievement of bone union for all treatments, but not in the negative control (
(340) Micro-CT Quantification of the percentage bone volume of total volume (BV/TV) for the treatment groups after weeks 4 and 8 confirmed the effects of the HS3 alone were more than comparable with FDA-approved BMP2 (
(341) Torsional Testing. After sacrifice at 8 weeks post-surgery, the rabbit ulnas were retrieved, wrapped in PBS-soaked gauze to maintain moisture, and frozen at 20 C. until torsional testing. Upon thawing, the ulna ends were potted in polymethylmethacrylate (Meliodent Rapid Repair, Heraeus Kulzer), contained within customized plastic blocks and allowed to solidify, to enable stable fixation. Ulnas were subsequently mounted in a MTS 858 Mini Bionix II testing system (MTS, Eden Prairie, Minn.). Polymer blocks and gauze were gently removed prior to testing. Each specimen was then tested to failure in torsion and the resulting torqueangular displacement curves were recorded. The rotation rate used was 1 degree per second until 35 degrees was reached and data were collected at 100 Hz. The stiffness, maximum torque, and angle at failure were recorded for each specimen, with the stiffness being measured as the slope of the linear portion of the torqueangular displacement curve (M. Bostrom, J. M. Lane, E. Tomin, M. Browne, W. Berbenan, T. Turek, J. Smith, J. Wozney, T. Schildhauer. Use of bone morphogenetic protein-2 in the rabbit ulnar non-union model. Clin Orthop Relat Res 1996; 327: 272-282). Statistical Analyses. Quantitative data was obtained in triplicates and reported as meansstandard deviation. Statistical analyses were performed using the Student's t-test (GraphPad software), and a p-value of less than 0.05 was considered significant.
(342) Quantification of stiffness and maximum torque assessed from control, BMP2 and HS-treated ulnae. Both stiffness and maximum torque was significantly improved for the treatment groups. Remarkably, the HS3-alone treatment resulted in mechanical properties that were similar to BMP2 treatment and intact bone at week 8 (