Bisphosphonate-Linked Hyaluronic Acid Hydrogel Sequesters and

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Bisphosphonate-linked hyaluronic acid hydrogel sequesters and enzymatically releases active bone morphogenetic protein-2 for induction of osteogenic differentiation Gry Hulsart-Billström, Pik Kwan Yuen, Richard Marsell, Jöns Hilborn, Sune Larsson, and Dmitri A. Ossipov Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/bm400639e • Publication Date (Web): 15 Aug 2013 Downloaded from http://pubs.acs.org on August 23, 2013

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Bisphosphonate-linked hyaluronic acid hydrogel sequesters and enzymatically releases active bone morphogenetic protein-2 for induction of osteogenic differentiation Gry Hulsart-Billström1, Pik Kwan Yuen1, Richard Marsell1, Jöns Hilborn2, Sune Larsson1, and Dmitri Ossipov2*. 1. Department of Surgical Sciences, Orthopedics, Uppsala University Hospital, Uppsala, SE 751 85, Sweden, 2. Science for Life Laboratory, Division of Polymer Chemistry, Department of Chemistry-Ångström, Uppsala University, Uppsala, SE 751 21, Sweden KEYWORDS: hyaluronic acid, bisphosphonate, hydrogel, BMP-2, enzymatic release, osteoinduction. ABSTRACT: Regeneration of bone by delivery of bone morphogenetic proteins (BMPs) from implantable scaffolds is a promising alternative to the existing autologous bone grafting procedures. Hydrogels are used extensively in biomaterials as delivery systems for different growth factors. However, a controlled release of the growth factors is necessary to induce bone formation, which can be accomplished by various chemical functionalities. Herein we demonstrate that functionalization of a hyaluronan (HA) hydrogel with covalently linked

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bisphosphonate (BP) ligands provides efficient sequestering of BMP-2 in the resulting HA-BP hydrogel. The HA-BP hydrogel was investigated in comparison with its analog lacking BP groups (HA hydrogel). While HA hydrogel released 100% of BMP-2 over two weeks, less than 10% of BMP-2 was released from the HA-BP hydrogel for the same time. We demonstrate that the sequestered growth factor can still be released by enzymatic degradation of the HA-BP hydrogel. Most importantly, entrapment of BMP-2 in HA-BP hydrogel preserves the growth factor bioactivity which was confirmed by induction of osteogenic differentiation of mesenchymal stem cells (MSCs) after the cells incubation with the enzymatic digest of the hydrogel. At the same time, the hydrogels degradation products were not toxic to MSCs and osteoblasts. Furthermore, BP-functionalization of HA hydrogels promotes adhesion of the cells to the surface of HA hydrogel. Altogether, the present findings indicate that covalent grafting of HA hydrogel with BP groups can alter the clinical effects of BMPs in bone tissue regeneration. INTRODUCTION Under normal conditions, bone is one of few tissues, which can heal without forming scar tissue. However, large bone defects following trauma are notoriously known to be associated with impaired healing as the basic biological requirements for successful bone regeneration, such as osteogenic cell migration and proliferation, osteoconductive matrix production, growth factor production, and neoangiogenesis are insufficient to form enough bone.1, 2 In these cases, the use of autologous bone grafting in an attempt to reconstitute normal fracture healing and hereby achieve union is still considered as a gold standard. Autograft usually works well although the method is associated with several drawbacks such as donor site morbidity, infection, pain, long surgery time as well as variability in the bone inductive capacity of the grafted bone which makes the method unpredictable.3


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As an alternative to bone grafting, recombinant human bone morphogenetic proteins (BMP-2 and BMP-7) are therefore used. BMPs belong to the transforming growth factor β-super family. The secreted protein binds to BMP receptors activating intracellular SMAD proteins, which translocate into the nucleus to regulate transcription of osteogenic factors. BMP-2 is normally expressed early in the bone-healing cascade and it is crucial for fracture healing.1, 4, 5 However, although the use of BMP-2 enhances fracture healing, its bone inducing potential has not yet been reached due to several reasons. Instability and short half-life of BMP-2 in vivo force to load supra-physiological doses of BMP-2 into current scaffolds. The use of BMP-2 at high doses might lead to side effects such as inflammation and tumor development. There is also a risk of ectopic bone formation when handling the scaffolds with high dosage of BMP-2. Not needless to mention is that BMPs are expensive compounds. If doses can be reduced while bone inductive capacity on the same time is being preserved, it will reduce the cost per treatment. To overcome some of the shortcomings of the present use of BMPs, there is a need for scaffolds that can effectively sequester the growth factor at the site of administration, protect it against degradation and/or aggregation and optimally deliver the growth factor to the cells at physiological doses. Preferably, these novel scaffolds should present better handling properties (injectability) to exclude open surgery as well as they should not elicit an immunogenic response.6 Competent offthe-shelf biomaterials that deliver active BMPs in a sustained manner during the time of healing should enhance therapeutic potential of the proteins in clinical orthopedics.4, 7 Hyaluronic acid (HA) is a linear polysaccharide with repetitive D-glucuronic acid and Nacetyl-D-glucosamine monosaccharide. In physiological solutions, HA macromolecules adopt stiff helical configuration with a coil structure of large hydrodynamic volume. Due to extensive entanglements of HA macromolecules, a physical network is formed functioning as a barrier against rapid transport of plasma proteins, thus providing their immobilization. High levels of


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endogenous HA are detected in remodeling and healing tissues with high cell proliferation and migration. HA promotes cell migration by interacting with cell surface receptors.8,

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exogenous HA failed to enhance fracture healing because of its rapid turnover. However, modified HA with lower turnover rates has shown promising results in regeneration of bone.10-16 HA hydrogels have been investigated as carriers for growth factors to enhance tissue healing, exhibiting non-immunogenic properties17 and in vivo degradability. Moreover, it has been demonstrated that HA hydrogels can retain BMP-2.16, 18, 19 HA has also been found to affect the interplay between osteoclasts and osteoblasts that play an important role in bone remodeling and fracture healing.20 These properties make modified HA hydrogels potential ideal candidates as scaffolds for BMPs in bone healing applications. Bisphosphonates (BPs) are the analogues of pyrophosphate in which the bridging oxygen is substituted by carbon.21 In contrast to organic phosphates, the P−C−P bridge of BPs is hydrolytically stable22 and provides synthetic possibilities to link BPs to other molecules. BPs have exceptionally high affinity to calcium ions as well as to the bone mineral hydroxyapatite.23 After oral or intravenous administration, BPs are targeted to bone tissue where they remain over an extended period. Most common clinical use of BPs is for the treatment of osteoporosis and osteolytic bone diseases (Paget’s disease and hypercalcemia) due to chemisorption of BPs to bone mineral followed by inhibitory effect on bone-resorbing cells, osteoclasts.24 Aminobisphosphonates such as pamidronate inhibit an enzyme called farnesyl pyrophosphate synthase, which impairs important signaling of GTPases that regulates a variety of cell functions, leading to apoptosis of osteoclasts.25 It was long thought that BPs would only slow down bone repair since they disrupt an array of cross-talk networks between the osteoclasts and the boneproducing cells, osteoblasts, thus decreasing bone formation.26 However, very recently it has


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been shown that, in bone repair, osteoblasts can work independently and anti-osteoclast activity of BPs may have a net anabolic effect when BPs are combined with scaffolds.27 This work is a part of our ongoing studies aimed at the exploration of the potential of HA hydrogel with BPs covalently attached to the matrix in bone tissue engineering. The main rational of attachment of BP ligands to HA hydrogels was threefold. First, strong binding affinity of BPs to calcium ions permits efficient mineralization of the BP-linked hydrogel, which has been demonstrated by us previously through incubation of the HA-BP hydrogel in a medium containing soluble calcium and phosphate ions.28 This implies the potential of the material for bio-mineralization in vivo, which is one of the factors of bone formation. Secondly, the degradation products of HA-BP hydrogel could potentially act on osteoclasts, similarly to the conventional unlinked BP drugs. In this way, the degradation products may prevent bone resorption. The effect of HA-BP conjugates on bone homeostasis is under current investigation and will be reported elsewhere. Third and the main question that was addressed in this study, was the effect of BPs on retention and release of BMP-2 when covalently immobilized to a HA matrix. It is expected that BMP-2 with isoelectric point around 9 should be non-specifically bound to negatively charged BP groups. More negatively charged HA-BP hydrogels should act similarly to sulfated glycosaminoglycans such as heparin or heparan sulfate. Bhakta et al., for example, improved BMP-2 retention properties of glycosaminoglycan hydrogels by introducing heparin.29 Moreover, modification of HA hydrogel with Ca2+-binding groups could provide additional Ca2+-mediated linkages with the growth factor. Calcium phosphate nano-phases of the mineralized hydrogel can also act as anchoring sites for the attachment of the cells surrounding the implanted hydrogel material. We, therefore, studied the attachment of rat mesenchymal stem cells (MSCs) and osteoblast-like cells on the surface of HA-BP hydrogel in comparison with a control hydrogel lacking BP groups (HA hydrogel). Finally, we investigated whether the


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attachment of BPs to HA can compromise biological safety of HA materials by conducting cytotoxicity studies with the HA-BP hydrogel degradation products. MATERIALS AND METHODS Preparation of hydrogels. HA-aldehyde (HA-al) was dissolved in PBS (Dulbecco’s Phosphate Buffered Saline, Sigma-Aldrich Inc., St Louis, MO, US) at 20 mg/mL concentration, while HAhydrazide (HA-hy) and dually modified HA-BP-hydrazide (HA-BP-hy) were dissolved at 25 mg/mL concentration. Formulation buffer (0.07% L-glutamic acid, 2.5% glycine, 0.5% sucrose, 0.029% sodium chloride, and 0,01% polysorbate, all purchased from Sigma-Aldrich Inc., St Louis, MO, US) was used to reconstitute BMP-2 from InductOS® (Wyeth, Madison, NJ) according to the manufacturer’s protocol. Aliquots from either stock 1.5 mg/mL or diluted 50 µg/mL solutions of BMP-2 were added to 25 mg/mL solutions of HA-hy or HA-BP-hy which resulted in 20 mg/mL solutions of the hydrazide-modified HA components containing BMP-2. Equal volumes of the HA-aldehyde solution and the BMP-2 containing solution of hydrazidemodified HA component (HA-hy or HA-BP-hy) were mixed to give hydrogel-forming formulations of BMP-2 with final concentrations of 5 or 150 µg/mL. After mixing, 120 µL of the combined solutions (HA-al + HA-hy or HA-al + HA-hy-BP) were immediately transferred to 2 mL low binding Eppendorf tubes. The self-standing hydrogels (HA hydrogel by mixing of HA-al and HA-hy derivatives and HA-BP hydrogel by mixing of HA-al and HA-hy-BP derivatives) were formed within 30 sec after mixing but were kept for complete cross-linking in the low binding Eppendorf tubes at 22°C overnight. In vitro release of BMP-2 from the hydrogels. 0.5 mL of supplemented release medium was added to the hydrogel samples. After certain time points (1, 3, 6, 12, 24, 48, 72 hours, and days 6, 9, 12, 14) the release medium was separated from the hydrogel samples, transferred to low


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binding tubes and stored at -80°C for later analysis by ELISA using human BMP-2 ELISA Development kit (PeproTech Inc, Rocky Hill, NJ). Three replicates were prepared for each group (control HA and HA-BP hydrogels). BMP-2 from the same source (InductOs®, Wyeth, Madison, NJ) was used to obtain a standard calibration curve and quantify the amount of BMP-2 released. Particularly, BMP-2 from Wyeth was calibrated in a diluent (0.05% Tween-20, 0.1% BSA in 1 × PBS) to give standard solutions with concentrations ranging between 0.125 and 8 ng/mL. The standard solutions were analyzed by ELISA according to the manufacturer’s protocol. Absorbance of solutions in micro-plates was measured at 405 nm with correction at 650 nm (Multiskan EX Microplate Photometer, Thermo Scientific, Waltham, MA, US). To take into account possible deactivation of BMP-2 in the release medium, 5 or 150 µg/ml of BMP-2 was incubated in the release medium for a specified period of time t equal to the release time from the hydrogels. After this time, the amount of BMP-2 in the medium was assessed by ELISA and considered as M

active

(t). Percentage of the released protein was then calculated as M

released

(t) / M active (t) × 100%. in place of M released (t) / M0 × 100% (M0 is the amount of loaded BMP-2 and is equal to 5 µg/ml × 0.2 mL = 1 µg or 150 µg/ml × 0.2 mL = 30 µg). While M0 is a constant, M active (t) depends on time and gradually becomes less than M0 over the time. Hydrogel degradation and determination of amount of BMP-2 among the degraded products. 1 mg of hyaluronidase (Hase, Sigma-Aldrich Inc., St Louis, MO, US) was dissolved in 1440 mL of PBS. 360 µL of the obtained Hase solution was added to each hydrogel sample after the release studies, and the hydrogels were kept overnight till complete dissolution. The solutions obtained after degradation of the hydrogels were analyzed by ELISA (PeproTech Inc, Rocky Hill, NJ. US). ELISA quantification of BMP-2 incubated with soluble HA derivatives. In parallel to the release studies, 11.75 µL of BMP-2 solution (50 µg/mL) was combined with 108.25 µL of PBS


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buffer containing 10 mg/mL HA-BP-hy or HA-hy derivative to give finally the solution of BMP2 with concentration 5 µg/mL. The resulting solution was mixed with 0.5 mL of either cell culture medium or PBS. The mixtures were incubated for 24 hours at 37ºC and then transferred to low binding tubes. The collected samples were kept at -80°C before analysis by ELISA. In the control experiments, BMP-2 formulations of the same final concentration were prepared in either cell culture medium or PBS. BMP-2 was detected by human BMP-2 ELISA Development kit (PeproTech Inc, Rocky Hill, NJ. US). Osteogenic activity of BMP-2 retained in the hydrogels. After completion of the release experiments, the hydrogels (120 µL by volume) were incubated overnight with 360 µL of Hase solution (695 U/mL) at 37°C. For comparison, similar hydrogels without BMP-2 were prepared and then degraded with hyaluronidase. 20 µL of the hydrogel-derived solutions obtained after degradation were mixed with 180 µL of culture medium and transferred to 96 well plates that were pre-seeded with rat bone marrow stromal cells (W-20 clone 17, ATCC, Teddington, UK) in α-MEM (supplemented with 2 mM L-glutamine, 1% (v/v) penicillin-streptomycin (both SVA, Uppsala, Sweden), and 10% (v/v) fetal bovine serum (Sigma-Aldrich Inc., St Louis, MO, US); 5% carbon dioxide, 95% air, 100% humidity) at 2000 cells/well density and 37°C. The hydrogelderived solutions of HA-BP hydrogel were diluted 10 times while the HA hydrogel derived solutions was taken without dilution and were added to the cells. The dilution factor was calculated basing on the difference in concentration of BMP-2 measured for the hydrogels digests by ELISA. The cells were cultured with digests for 48 hours and then washed with PBS. The cells were lyzed in 70 µL of sterile water with two freeze-thaw cycles at -80°C and 37°C. ALP activity was measured by incubating 50 µL of the cell lysate with 100 µL of Alkaline Phosphatase Yellow pNPP Liquid Substrate (Sigma-Aldrich Inc., St Louis, MO, US) at 37°C for


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30 minutes. Absorbance was measured at 405 nm wavelength (Multiskan EX Microplate Photometer, Thermo Scientific, Waltham, MA, USA). Cell attachment and viability on the surfaces of hydrogels. Hydrogels of 60 µL volume were formed in 96 well plate with 5 repeats for each type of hydrogels. Rat osteoblasts-like cells (UMR ROS-106) and rat MSCs (RMSC R492-05) (both from Health Protection Agency Culture Collections, Salisbury, UK) were seeded on the hydrogels at a density of 1000 cells/well and incubated for 4 hours at 37 °C and 5% CO2 after which the unattached cells were washed off with PBS. For a control, the cells were also seeded on a tissue culture plastic. The attached cells were analyzed by adding of 20 µL MTS solution (Promega, Madison WI, US) to each well and incubating for 4 hours at 37° C in darkness. UV absorbance of the formazan product was measured at 492 nm (Multiskan EX Microplate Photometer, Thermo Scientific, Waltham, MA, US). Cytotoxicity of the hydrogels degradation products. Hydrogels without BMP-2 were formed as was described above, except that hydrazide components of HA (HA-hy and HA-BP-hy) were initially dissolved in PBS at 20 mg/mL concentration. The hydrogels were kept overnight at room temperature for complete setting. 2 mg of hyaluronidase (Hase, Sigma-Aldrich Inc., St Louis, MO, US) was dissolved in 2000 mL of PBS. 300 µL of the Hase solution was added to each hydrogel sample of 300 µL volume and the hydrogels were incubated overnight at room temperature till complete dissolution. The hydrogels digests were heated at 90°C for 5 min to inactivate the Hase. The heat-treated digests were then freeze-dried and finally stored at 2-8°C. The freeze-dried digests were re-dissolved in cell culture medium α-MEM (supplemented with 2 mM L-glutamine, 1% (v/v) penicillin-streptomycin (both SVA, Uppsala, Sweden), and 10% (v/v) fetal bovine serum (Sigma-Aldrich Inc., St Louis, MO, US) and diluted to three


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concentrations: 0.5%, 0.05%, and 0.005%. To sterilize the dissolved digest, the solutions were heated to 95°C, after which it was cooled down, and added to the cells. The CellTiter 96® Aqueous Non-Radioactive Cell Proliferation Assay from Promega, Madison, WI, US was used to determine the viability of cells. Osteoblasts-like cells (UMR ROS106) and MSCs (RMSC R492-05) (both from Health Protection Agency Culture Collections, Salisbury, UK) were seeded on a 96 culture well plate (CoStar®, Corning Incorporated, Corning, NY, US) at concentration of 4000 cells/well. When 50% of confluence was reached, the digests (100 µL) were added and incubated at 37°C. After 36 hours of incubation, MTS reagent was added to the wells and UV absorbance of the formazan product was measured at 492 nm (Multiskan EX Microplate Photometer, Thermo Scientific, Waltham, MA, US). RESULTS AND DISCUSSION In vitro release of BMP-2 encapsulated in hydrazone cross-linked HA hydrogels. HA derivatives with hydrazide-terminated groups (HA-hy) were used for hydrazone networking reaction upon mixing with the aldehyde-modified HA (HA-al). To allow “click” modular assembly of hydrogels with covalently attached BP ligands and at the same time to provide mild encapsulation of a growth factor (BMP-2), HA was dually functionalized with orthogonal chemoselective groups (i.e., thiol and hydrazide) to give HA-SH-hy (Figure 1).30 Thiol groups of HA-SH-hy were utilized for the attachment of acrylated BP derivative, while the remaining hydrazide groups allowed “click” conversion of the grafted HA derivative (HA-BP-hy) into the subsequent HA-BP hydrogel (Supporting Information, Figure S1). The BP-linked hydrogel was directly compared to the control hydrogel that was formed by mixing HA-hy derivative with HAal and therefore did not contain BP groups. Degree of functionalization with hydrazide groups in HA-hy and HA-BP-hy derivatives was the same (10% of the total amount of HA disaccharides)


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to maintain the same cross-linking density in the resulting hydrogels. This allowed measuring the effect of BP ligands on the release of BMP-2 from the hydrogels, on adhesion of stem cells to the surface of the hydrogels, and on viability of the cells towards the hydrogel degradation products. The synthesis of all hydrogel-forming HA derivatives have been described in our previous publication.28 Mixing induced two-component hydrogels were formed upon combination of a hydrazide component (HA-hy or HA-BP-hy) with the aldehyde derivatized HA (HA-al).28 Mechanical properties of the hydrogels (thereafter named as HA and HA-BP hydrogels) were studied after the hydrogels formation followed by setting for 24 hours. Apparently, elastic modulus G′ of HABP hydrogel was 1.7 times less as compared to HA hydrogel (Table S1) which should be attributed to the increased repulsive electrostatic interactions between pendant BP groups of the HA-BP hydrogel. This subsequently led to the increased swelling of HA-BP hydrogel in PBS (Table S1). BMP-2 was in situ encapsulated in the hydrogels by addition of the growth factor to the solutions of hydrazide components (HA-hy or HA-BP-hy) at two concentrations. The final concentration of BMP-2 in the hydrogels was either 5 or 150 µg/mL, which resulted in four groups of the hydrogel samples: low dose HA, low dose HA-BP, high dose HA, and high dose HA-BP. After setting for 24 hours, the hydrogels were incubated in cell culture medium, which was replaced with fresh medium regularly after certain intervals of time. The concentration of BMP-2 was determined in the collected samples of the release medium by enzyme-linked immunosorbent assay (ELISA). The release experiment revealed drastic differences in BMP-2 retention properties of the studied hydrogels. We found that BMP-2 was almost completely retained in HA-BP hydrogel as compared with the control HA hydrogel. Particularly, the highest amount of BMP-2 was released from the high dose HA hydrogel (150 µg/mL of BMP-2) already after 3 hours of the hydrogel incubation and reached 7.82 ± 0.89 µg/mL (Figure 2a). For


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comparison, the highest amount of BMP-2 released from the analogous high dose HA-BP hydrogel was 0.505 ± 0.036 µg/mL after 12 hours of the hydrogel incubation. Almost the whole amount of loaded BMP-2 was released from the high dose HA hydrogel during the first 5 days with only minor amounts of BMP-2 being released during the remaining time of the release experiment (days 6 to 14) (Figure 2b). In contrast, the release of BMP-2 from the high dose HABP hydrogel was much more gradual with small amounts of detectable BMP-2 during the entire study period. In total, only 5% of the whole amount of loaded BMP-2 was released from the high dose HA-BP hydrogel. In contrast to HA hydrogel, BMP-2 could still be detected in the release medium of HA-BP hydrogel during the last five days of the release experiment (three time points at days 9, 11 and 14), indicating large amounts of the retained BMP-2 in this hydrogel. The same trend was also observed for the low dose hydrogels (5 µg/mL of BMP-2, Figure 2 c, d). Liberation of BMP-2 from HA hydrogels by enzymatic degradation. The decreased amount of BMP-2, detected by ELISA in the released medium of HA-BP hydrogel, can be a result of growth factor retention in the hydrogel as well as due to the growth factor being unavailable for the antibody for some unknown reasons (degradation, aggregation, side-binding to lab ware). To figure out, whether BMP-2 was retained in the hydrogel or not, the HA-BP and HA hydrogels were degraded with hyaluronidase (Hase) after the release study. The obtained enzymatic digests were subsequently analyzed by ELISA (Figure 3). This experiment revealed that large amount of BMP-2 was still present in the degraded HA-BP hydrogels. Specifically, high dose HA-BP hydrogels of 200 µL volume were loaded with 18 µg of BMP-2, they released 0.9 ± 0.02 µg of the protein during the release study, and 2.7 ± 0.2 µg of BMP-2 was detected in the hydrogel digest after the release study (Figure 3a). It accounts for 15.8 % of the theoretical amount of BMP-2 that should retain in the high dose HA-BP hydrogel after the release study. 0.33 ± 0.03


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µg of BMP-2 was detected in the digest of high dose HA hydrogel which is more than 8 times less than the actual amount of BMP-2 detected in the digest of the high dose HA-BP hydrogel. This difference was even higher (almost 20 times) for the low dose hydrogels (Figure 3b). It should be noted that sufficiently higher percentage (39.7 %) from the theoretical amount of the retained BMP-2 were actually detected in the digests of low dose HA-BP hydrogels. They were initially loaded with 0.6 µg of BMP-2, they released 0.036 ± 0.003 µg of the protein during the release study, and 0.224 ± 0.02 µg of BMP-2 was detected in the hydrogel digest after the release study (Figure 3b). These results indeed confirmed the very high binding ability of the HA-BP hydrogel toward the encapsulated growth factor at both high (150 µg/mL) and low (5 µg/mL) BMP-2 loading. A profound retention of BMP-2 in HA-BP hydrogel compared to regular HA hydrogel can be on one side rationalized in terms of electrostatic interactions. BMP-2 has pI of 9 meaning that it will be retained in the hydrogels formed by polyanionic HA chains. BP groups are completely ionized at physiologic conditions and bear more negative charge as compared to the carboxylate groups of HA. By grafting of BP groups to HA matrix, one can therefore expect enhanced electrostatic interactions in accordance to the reported effect of the sulfated GAGs (heparin). Bhakta et al. showed a release of BMP-2 of around 50% when functionalizing a cross-linked HA hydrogel with heparin.29 HA hydrogel without immobilized heparin released 70% of BMP-2 at the same time point. In the present study, the difference in BMP-2 release was so pronounced that it can hardly be explained by only electrostatic forces. Another possible interaction between BMP-2 and BP groups can be through chelating to metal ions such as Ca2+ and Mg2+. Absorption of BMP-2 on hydroxyapatite (HAP) surface has been reported to involve essential interactions between Ca2+ ions from the surface and carboxylic groups from the protein.31 The release of the


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growth factor from calcium phosphate ceramics was rather low and reached only 23% after 21 days. In other studies, favorable binding of BMP-2 to HAP in presence of Ca2+ ions was observed since Ca2+ ions are absorbed on phosphate sites of HAP surface and to the carboxylic of acidic residues of BMP-2.32, 33 The same Ca2+-bridging effect might be expected between BP groups of the matrix and the chelating groups of BMP-2. In the present study, the release experiments were performed in αMEM medium containing Ca2+ ions which provided mineralization of the HA-BP hydrogel, but not the control HA hydrogel, according to our previous observation.28 BMP-2 may bind to the mineral nano-phases of the HA-BP matrix in the same way as to macroscopic HAP surface, i.e. through Ca2+ ions. This type of chemical bonding is very strong and thus desorption appeared clearly as the limiting step of the release. Hence, the release of most of the growth factor necessitated a degradation of the hydrogel. Such process occur in vivo suggesting enzymatic release of the growth factor controlled by surrounding cells. HA-BP derivatives can stabilize BMP-2 against de-activation through binding to the growth factor. Strikingly slower release from the HA-BP hydrogel as compared to regular HA hydrogel can only be attributed to the presence of the matrix-appended BP groups. The degradation of HA-BP hydrogel results in generation of soluble heterogeneous BP-grafted fragments of HA macromolecules of different molecular weight. The very strong electrostatic as well as Ca2+-mediated binding of BP groups with the chelating side chains of the growth factor, existing in the HA matrix, should therefore be present even after enzymatic degradation of HABP hydrogel. Hence, BMP-2 is likely to be bound to the HA degradation products through BP groups. It was important to verify whether the soluble HA-BP•BMP-2 complex can be detected by ELISA as free BMP-2 (Figure 4). ELISA was used for quantification of BMP-2 when incubated in PBS and in cell culture medium together with either soluble HA-BP-hy or HA-hy


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derivative and the results were compared with a setting when the same amount of free BMP-2 was incubated in pure media. Interestingly, the amount of the detected BMP-2 after incubation with HA-BP-hy was more than the amount of the detected protein after its incubation alone (control medium in Figure 4). Oppositely, incubation of BMP-2 with HA-hy derivative resulted in the decrease of the detectable protein. These results demonstrated at least that binding of BMP-2 to HA-BP-hy does not block the epitopes recognized by BMP-2 specific antibody. We found also that the effect of HA-BP-hy on BMP-2 was more pronounced in PBS than in culture medium. The amount of detectable BMP-2 was three times higher for the BMP-2•BP-HA-hy complex than for free BMP-2 after 24 hours of incubation in PBS (Figure 4). BMP-2 is known to be very unstable under neutral pH (PBS) with tendency to aggregation.19, 34

Aggregation of BMP-2 lowers the amount of therapeutically effective growth factor

significantly. The results presented here can be explained by that BMP-2 is most likely protected against de-activation in the complex with BP-functionalized HA. It was noteworthy that the attached BP groups were crucial in stabilizing the growth factor, since the HA derivative lacking BP groups (HA-hy) did not have such an effect both in culture medium as well as in PBS. Stabilization of BMP-2 can be achieved upon Ca2+-mediated coating of the growth factor with HA-BP derivatives. BMP-2 contains calcium-binding sites and the presence of the ions together with strongly chelating BP ligands may affect the conformational stability of the protein and consequently its activity profile. Other studies have also shown that proteins incorporated into a polymer matrix remain active after mineralization.35 BMP-2 enzymatically liberated from HA-BP hydrogel induces osteogenic differentiation of MSCs. ELISA can only provide the information about availability of functional structural epitopes of a growth factor. It does not tell about its bio-functionality, which can only be assessed in a cellular assay. Alkaline phosphatase (ALP) is a membrane bound enzyme that is


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often used as a marker for osteogenic differentiation. Therefore, mesenchymal stem cells (MSCs) were incubated with the digests obtained after the hydrogel degradation. To rule out that the hydrogel compounds alone do not have an effect on ALP activity, we have also prepared hydrogels without BMP-2, degraded them, as it was done with BMP-2-loaded hydrogels and the degradation products were added to the cells. After 48 hours of incubation, ALP activity of the cells was measured by spectrophotometric monitoring the conversion of an ALP substrate, pnitrophenol phosphate, into p-nitrophenol (Figure 5). It can be seen that ALP activity of the cells alone (negative control in Figure 5) is similar to the activity of the cells cultured with the growth factor-free hydrogel degradation products. Similarly, ALP activity of the cells treated with BMP2-loaded HA hydrogel was close to the negative control. However, treatment of the cells with the digests of BMP-2-loaded HA-BP hydrogel resulted in higher ALP activity of the cells. It should be noted that the digests of HA-BP hydrogel were diluted 10 times while the HA hydrogel digest was taken without dilution and added to the cells to adjust the concentration of BMP-2 to the same level (approximately 75 ng/mL). This has been done basing on the difference in concentration of BMP-2 that was assessed for the hydrogels digests by ELISA (see Figure 3 showing that almost the total amount of BMP-2 had been released from HA hydrogel and only minor amounts of BMP-2 were detected in the hydrogel after its degradation). These results confirmed our assumption about a protective environment being provided to BMP-2 by the BPmodified matrix and the soluble HA-BP derivatives. The higher amount of the growth factor was subsequently translated into a more effective signaling cascade with ultimate higher production of ALP by the cells. The results of this ALP assay experiments inevitably showed that BMP-2 entrapped in the HA-BP hydrogel can be thus protected against de-activation for many days. The growth factor can however be released enzymatically from the hydrogel in the form of complex


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with HA-BP degradation products that preserves the growth factor activity for some time in physiological solution. HA-BP hydrogel is adhesive towards progenitor bone cells and to osteoblasts. Our noncellular approach to bone regeneration is based on effective delivery of growth factors to endogenous stem cells as well as on recruitment of progenitor cells actively into the regeneration process. If the implanted hydrogel material is adhesive to the stem cells, this will allow the recruited cells to attach to the surface followed by infiltration into the hydrogel, ultimately leading to more efficient and faster formation of bone tissue. Therefore, important characteristic of a new biomaterial is its interface with the cells on which it should act. We have therefore evaluated the adherence of both rat MSCs and osteoblasts towards the surfaces of HA-BP and HA hydrogels. More cells remained attached to the surface of HA-BP hydrogel (Figure 6). This observation was in accordance with other studies with synthetic phosphonic-acid containing hydrogels. The adhesion and proliferation of osteoblast-like MG-63 cells was significantly improved on hydrogels with increasing content of polymerized vinyl phosphonic acid.36 The idea that phosphonate-containing polymers interact with cell-adhesive, naturally occurring proteins has been exploited in a series of other studies.37,

38

We believe that the

mechanism that retain the growth factor in HA-BP hydrogel should also be in action with cellular membrane proteins. BP groups on the surface of the hydrogel attract Ca2+ ions from the medium, which is followed by deposition of hydroxyapatite-like calcium phosphate phase.39 The Ca2+ ions then can act as “bridges” between chelating groups of the membrane proteins and either phosphate or BP groups on the hydrogel surface. The increased adhesion of the cells may also result in promotion of the cellular ALP activity, even without the presence of a direct chemical signal (BMP-2).


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Cytotoxicity of HA-BP hydrogel degradation products. We have shown previously that hydrazone cross-linked HA hydrogels are not toxic to cells by conducting in vitro 2-D and 3-D experiments.10,11,18 In vivo HA hydrogels undergo enzymatic degradation with the liberation of HA macromolecules of different molecular weight. Degradation of BP-modified HA hydrogel will therefore result in different soluble species of HA chains with the pendant BP functionality. Hence, it is particularly important for the bone regeneration application to assess the cytotoxicity of potential degradation products of a hydrogel biomaterial. In this study, we used MTS assay to investigate the cytotoxicity of the degraded HA-BP hydrogel. The hydrogels were degraded using 300 U/mL hyaluronidase and the resulting digests added to rat MSCs and rat osteoblastlike cells at three concentrations: 0.5%, 0.05%, and 0.005%. Interestingly, MSCs showed increased cell viability in response to increased concentration of the HA degradation products with highest cell viability at 0.5% (Figure 7a, b, c). Moreover, they were more viable than the control cells not treated with the degraded HA. This can be explained by the fact that HA receptors are found on the surface of MSCs that are recruited to the sites where tissue healing takes place.40, 41 High concentrations of HA are detected in tissues with high cell proliferation and where cell migration takes place.9 It is noteworthy, that the BP-linked HA and HA lacking BP groups resulted in almost the same MSCs viability. The viability of osteoblasts was 100% when treated with 0.005% of HA degradation products, i.e. the same as viability of the untreated osteoblast-like cells (Figure 7d). In contrast, viability of the osteoblast-like cells was reduced upon increasing the concentration of the digests to 0.05% and 0.5% (Figure 7 e, f). It should be noted, however, that the level of toxicity against osteoblast was comparable for the HA and HABP degraded products. CONCLUSIONS


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In this study we have shown that by providing suitable chemical functionality (bisphosphonate groups) linked to hyaluronic acid hydrogels, one can (i) alter dramatically the release profile of bone morphogenetic protein-2 when it is loaded into the hydrogel, (ii) protect the protein against premature deactivation, as well as (iii) improve cell interactive properties of the hydrogel through promotion of adhesion of bone progenitor stem cells (hMSCs) and osteoblasts onto the gel. This could be of great practical and clinical importance where the highly controlled release of therapeutically effective minimal doses a growth factor may reduce the cost of the current medical treatments and prevent their side effects such as ectopic bone formation and immune response against BMP-2.


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FIGURES:

Figure 1. Preparation of HA-BP and HA hydrogels with encapsulated BMP-2 by mixing of the corresponding hydrazide-modified components (HA-BP-hy and HA-hy, respectively) with the HA-al cross-linker containing BMP-2. Hydrazone cross-links are presented as red circles and the thioether linkages formed by Michael addition reaction are shown as blue circles. The structures of all hydrogels-forming HA derivatives are shown on the right.


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Figure 2. The release of BMP-2 from high (150 µg/mL) and low (5 µg/mL) dose HA hydrogels (black) and HA-BP hydrogels (white). Mean concentration ± SD of BMP-2 in the samples of release medium obtained at different time points of incubation of the high dose hydrogels (a) and low dose hydrogels (c). The cumulative release profile of the entire BMP-2 from the high dose hydrogels (b) and the low dose hydrogels (d). Results are presented as mean ± SD (n = 3).


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Figure 3. Masses of BMP-2 detected in the degraded HA hydrogels (white bars) and HA-BP hydrogels (black bars). The hydrogels were initially loaded with either 150 µg/mL (a) or 5 µg/mL (b) of BMP-2, subsequently used in the release experiments, and finally degraded with hyaluronidase. Results are presented as mean ± SD (n = 3).


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Figure 4. Determination of BMP-2 by ELISA after 24 hours of incubation of the protein (patterned bars). The same amount of BMP-2 was also incubated together with either HA-BP-hy (black bars) or HA-hy (white bars) derivative. Incubation was performed either in culture medium or PBS. Results are presented as mean ± SD (n = 3).


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Figure 5. Cellular ALP activity of W20-17 rat stromal cells measured as absorbance of pnitrophenol at 405 nm and presented as mean ± SD (n = 7). The high dose hydrogels (150 µg/mL loading of BMP-2) or protein-free hydrogels were degraded with Hase. The degraded proteinloaded hydrogels were diluted with medium to contain approximately 75 ng/mL of BMP-2. The dilution factor for HA-BP hydrogel was 10 times higher than for HA hydrogel basing on ELISA determination of the recovered BMP-2 (see Figure 3). The cells were incubated with the diluted hydrogel digests for 48 hours. As a negative control, the cells were incubated with pure medium. For a positive control, the cells were incubated with medium containing 75 ng/mL of BMP-2.


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Figure 6. Adherence of rat MSCs and rat osteoblasts (UMR) on HA and HA-BP hydrogels. The cells were seeded on the hydrogels and washed after 4 hours of incubation. The attached cells were quantified by MTS assay and expressed as absorbance of the formazan product at 492 nm ± SD (n = 6). The adherence of the cells on tissue culture plastic (TCP) was measured for the control experiments.


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Figure 7. Cell viability of rat mesenchymal stem cells (MSCs) (a, b, c) and rat osteoblasts (UMR) (d, e, f) incubated with the hydrogels digests at 0.005% (a, d), 0.05% (b, e) and 0.5% (c, f) concentrations. The cells were seeded with the digests for 36 hours and the cell viability was measured by MTS assay. The viability of the cells on tissue culture plastic (TCP) was measured for the control experiments. The results are presented as the mean ± SD, n = 3.


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AUTHOR INFORMATION Corresponding Author *Dmitri Ossipov ([email protected]) Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ACKNOWLEDGMENT The research leading to these results has received funding from European Community’s Seventh Framework Programme (BIODESIGN). We would like to thank Britt-Marie Andersson for help and technical support and Inger Pihl Lundin for valuable advices in analytical detection of BMP2.


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For Table of Contents Use Only

Bisphosphonate-linked hyaluronic acid hydrogel sequesters and enzymatically releases active bone morphogenetic protein-2 for induction of osteogenic differentiation. Gry Hulsart-Billström1, Pik Kwan Yuen1, Richard Marsell1, Jöns Hilborn2, Sune Larsson1, and Dmitri Ossipov2*


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Bisphosphonate-Linked Hyaluronic Acid Hydrogel Sequesters and

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