Release kinetics of prolyl hydroxylase inhibitors from collagen barrier membranes.

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Article

Release kinetics of prolyl hydroxylase inhibitors from collagen barrier membranes

Journal of Biomaterials Applications 0(0) 1–9 ! The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0885328214556158 jba.sagepub.com

Omar Hamid1,2, Manuela Pensch1,2 and Hermann Agis2,3

Abstract Collagen barrier membranes are used in guided tissue regeneration to support healing. This strategy, however, relies on the healing capacity of the tissue. Pharmacological inhibitors of prolyl hydroxylases can support regeneration by enhancing angiogenesis and are therefore a promising tool for periodontology. Here we evaluate the release kinetics of the prolyl hydroxylase inhibitors dimethyloxalylglycine and L-mimosine from collagen barrier membranes. Dimethyloxalylglycine and L-mimosine were lyophilized onto the collagen barrier membranes. The morphology of the collagen barrier membranes was analysed using scanning electron microscopy. The release of prolyl hydroxylase inhibitors was assessed by colorimetric and spectroscopic methods. Their ability to induce a cellular response was assessed in bioassays with gingival and periodontal ligament fibroblasts based on vascular endothelial growth factor production, proliferation, and metabolic activity of the cells. We found that loading of collagen barrier membranes with prolyl hydroxylase inhibitors did not change the overall membrane morphology. Assessment of the release kinetics by direct measurements and based on vascular endothelial growth factor production showed that supernatants obtained from the collagen barrier membranes in the first 6 hours had a sufficient level of prolyl hydroxylase inhibitors to induce vascular endothelial growth factor production. A similar kinetic was found when cell proliferation was assessed. Changes in metabolic activity did not reach the level of significance in the MTT assay. In conclusion, collagen barrier membranes can release prolyl hydroxylase inhibitors thereby increasing the pro-angiogenic capacity of periodontal cells in vitro. These findings provide the basis for preclinical studies to evaluate the regenerative capacity of prolyl hydroxylase inhibitors in periodontology and oral surgery. Keywords Guided tissue regeneration, bone regeneration, material sciences, growth factors, biomaterials

Introduction In guided bone regeneration and guided tissue regeneration a barrier membrane is placed between the transplanted bone and the surrounding soft tissue to provide space maintenance and guide the healing process.1 Collagen barrier membranes are widely used due to their favourable properties.2–4 Collagen is biocompatible, degradable, and has been shown to support angiogenesis, making it an ideal candidate for scaffolds.2,5–7 Although biodegradability is important to prevent a second surgery, uncontrolled degradation and therefore lack of volume stability and loss of barrier function are undesired. Bi-layered membranes were therefore developed to enhance their stability and hinder cellular infiltration while providing a beneficial surface structure for the osteoblasts on one side and cells of the oral soft

tissue on the other side.7 However, the success of healing relies on the healing capacity of the tissue. Particularly when this healing capacity is compromised, regeneration of the alveolar bone and the periodontium represent a challenging situation. Therefore, there is a demand for strategies which support the healing

1 Department of Oral Surgery, Medical University of Vienna, Vienna, Austria 2 Department of Conservative Dentistry and Periodontology, Medical University of Vienna, Vienna, Austria 3 Austrian Cluster for Tissue Regeneration, Vienna, Austria

Corresponding author: Hermann Agis, Department of Conservative Dentistry and Periodontology, Medical University of Vienna, Sensengasse 2a, A-1090 Vienna, Austria. Email: [email protected]

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process. One approach is to combine the traditional concept of guided bone regeneration with pro-angiogenic growth factors.8 Although approaches using recombinant growth factors are effective, their biological stability in the catabolic defect environment and high price drives research for alternative strategies. The capacity of prolyl hydroxylase inhibitors to stimulate angiogenesis and thereby soft and hard tissue regeneration has drawn attention to these ‘hypoxia mimetic agents’. Prolyl hydroxylase inhibitors can stimulate tissue regeneration by stabilizing the label transcription factor hypoxia inducible factor-1alpha (HIF-1alpha).9,10 Animal studies revealed that by stabilizing HIF-1alpha prolyl hydroxylase inhibitors can help to overcome compromised wound healing.11–13 However, in these settings prolyl hydroxylase inhibitors were repeatedly injected into defect sites.9–13 In periodontal surgery regeneration a one-step strategy where prolyl hydroxylase inhibitors are released from a carrier into the defect to stimulate tissue regeneration would be desirable.14 Here we propose a concept where collagen barrier membranes are loaded with prolyl hydroxylase inhibitors to induce a pro-angiogenic response in the tissue, thereby utilizing a biomaterial as carrier for prolyl hydroxylase inhibitors that is already clinically applied. However, so far it is unclear if collagen barrier membranes can serve as carriers for these prolyl hydroxylase inhibitors and whether the molecules maintain their pro-angiogenic capacity when released. Before these membranes can be tested in preclinical models, in vitro studies are indicated that answer these questions and determine the release kinetics of the prolyl hydroxylase inhibitors. In the present study we assessed whether collagen barriers can induce a pro-angiogenic response in periodontal cells in vitro. We assessed the release kinetics of prolyl hydroxylase inhibitors utilizing direct measurement and established bioassays with gingival (GF) and periodontal ligament fibroblasts (PDLFs).15 The cellular response was determined based on vascular endothelial growth factor (VEGF) production, proliferation, and metabolic activity by immunoassays, 3[H]thymidine incorporation, and by 3-(4,5-dimethythiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) conversion, respectively. Thereby this study provides first insights into the feasibility of collagen barrier membranes as carriers for prolyl hydroxylase inhibitors.

The membranes were loaded with the prolyl hydroxylase inhibitors dimethyloxalylglycine (DMOG, Enzo Life Sciences, Lausen, Switzerland) and L-mimosine (L-MIM, Sigma) by lyophilisation. In detail, 50 ml of a 3 mM solution of the prolyl hydroxylase inhibitors in liquid form was added to collagen barrier membrane specimens of 5 mm diameter at room temperature. The samples were frozen at –80 C. Lyophilization was performed using a freeze dryer ALPHA 1-2 LDplus (Martin Christ, Osterode am Harz, Germany) over night, not exceeding 24 h.

Materials and methods Loading of collagen barrier membranes with prolyl hydroxylase inhibitors

Supernatants from the collagen barrier membranes were generated by adding 250 ml of serum free aminimal essential medium (Invitrogen Corporation, Carlsbad, CA) supplemented with antibiotics to one collagen barrier membrane specimen. Supernatants were collected and replaced with fresh medium at

Collagen barrier membranes (BioGideÕ ) were provided by Geistlich Pharma AG (Wolhusen, Switzerland).

Scanning electron microscopy (SEM) SEM imaging from the lyophilized collagen barrier membranes was generated utilizing the TM-1000 tabletop system (Hitachi, Tokyo, Japan). The collagen barrier membranes were mounted on an aluminium stub and images were taken from both sides of the membrane at 15,000 V accelerating voltage at a 200-fold magnification.

Release of prolyl hydroxylase inhibitors from the collagen barrier membranes for direct measurement of the prolyl hydroxylase inhibitors Supernatants from the collagen barrier membranes were generated by adding 250 ml of phosphate buffered saline (Invitrogen Corporation, Carlsbad, CA, USA) to one collagen barrier membrane specimen. Supernatants were collected and replaced with fresh medium at hour 1, 3, 6, 24, and 48. These supernatants were subject to direct measurement of the prolyl hydroxylase inhibitors. DMOG was measured as described previously with minor modifications using a DU530 life science UV/Vis spectrophotometer (Beckman Coulter, Inc., CA, USA) at 204 nm.16 L-MIM was measured using a colorimetric assay as described before.17 In brief, supernatants of the collagen barrier membranes were incubated with diazotized p-nitroaniline in a sodium phosphate buffer (pH 7.0) for 15 min and then measured in a Multiskan Ascent microplate reader (Thermo Labsystems, Waltham, MA, USA) at 405 nm. Quantification of prolyl hydroxylase inhibitors was performed using the standard curve method.

Release of prolyl hydroxylase inhibitors from the collagen barrier membranes for cell culture



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hour 1, 3, 6, 24, and 48. These supernatants were subject to bioassays with GF and PDLF.

(Peprotech) according to the description of the manufacturer.

Cell culture

Statistical analysis

After informed consent was obtained fibroblasts were prepared from extracted third molars (Ethics Committee of the Medical University Vienna, #631/2007). The donors had no previous history of dental inflammation. Fibroblasts were isolated as described previously.15 In brief GF were isolated scraping of the soft tissue particles of the gingiva from the tooth neck. These soft tissue particles were then expanded in vitro. PDLFs were isolated by scraping of the soft tissue from the tooth root and expanding it in vitro. GF and PDLF were cultivated in a-minimal essential medium (Invitrogen Corporation), supplemented with 10% fetal calf serum (PAA Laboratories, Linz, Austria) and antibiotics (Invitrogen Corporation) at 37 C, 5% CO2, and 95% humidity. Fibroblasts that had not undergone more than 10 passages were plated at 50,000 cells/cm2 in 96 well plates. Cells were incubated for 24 h with the supernatants from the collagen barrier membranes and were then subjected to viability and proliferation assays. Cell culture supernatants were assessed with VEGF immunoassays.

Data were compared by analysis of variance (ANOVA) and paired t-test. Significance was assigned at the p < 0.05 level.

MTT assay

Prolyl hydroxylase inhibitors are released from collagen barrier membranes within the first hours as indicated by direct measurement and increase in VEGF in fibroblasts from the gingiva and periodontal ligament

To measure metabolic activity, cells treated with supernatants of the collagen barrier membranes were incubated with 1 mg/ml MTT at 37 C for the last 2 h of culture. The MTT solution was removed and formazan crystals were solubilised with dimethyl sulfoxide. The optical density was measured with a photometer at 550 nm. Data are normalized to cells incubated with supernatants from untreated collagen barrier membranes. 3

[H]Thymidine incorporation assay

To measure proliferation, cells were pulse-labelled with 3 [H]thymidine (0.5 mCi/well, Hartmann Analytic, Braunschweig, Germany) for the last 6 h of exposure to the supernatants of collagen barrier membranes. The plates were subjected to liquid scintillation counting (Packard, Meriden, CT). Data were normalized to cells that were exposed to supernatants from unloaded collagen barrier membranes (controls).

Enzyme-linked immunosorbent assay for VEGF Conditioned medium from GF and PDLF that were exposed to supernatants from collagen barrier membranes were harvested and subject to enzyme-linked immunosorbent assay (ELISA) for VEGF. The measurements were performed using the VEGF ELISA kit

Results Loading of prolyl hydroxylase inhibitors onto collagen barrier membranes does not change their overall morphology To assess the morphology of the collagen barrier membranes SEM was performed. Overall lyophilisation of prolyl hydroxylase inhibitors onto the collagen barrier membranes did not change the morphology of membranes (Figure 1). The membranes showed the double-layered structure. One side of the membrane showed a smooth and compact structure consisting of mainly parallel fibres (Figure 1, smooth), whereas the other side of the membrane was rough and more porous (Figure 1, rough).

Direct measurements of the prolyl hydroxylase inhibitor in the supernatants of the collagen barrier membranes revealed that the majority of DMOG and L-MIM is released within the first hours with the highest levels in the first hour (Table 1). Bioassays with gingival and PDLFs were performed to assess the release kinetics of prolyl hydroxylase inhibitors from collagen barrier membranes (Figure 2(a) and (b)). We found that supernatants from all collagen barrier membranes loaded with prolyl hydroxylase inhibitors enhanced the VEGF production of gingival and periodontal fibroblasts. DMOG treated collagen barrier membranes induced a peak increase of 3.8-fold compared to the unloaded membranes in fibroblasts from the gingiva and 3.6-fold in fibroblasts from the periodontal ligament. L-MIM induced a similar response in VEGF production showing a 3.7- and 3.6-fold increase compared to unloaded membranes in gingival and periodontal fibroblasts, respectively. These data show that prolyl hydroxylase inhibitors loaded on collagen barrier membranes maintain their capacity to induce a pro-angiogenic response.



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DMOG

L-MIM

Rough

Smooth

Control

Figure 1. Scanning electron microscopy images of collagen barrier membranes loaded with prolyl hydroxylase inhibitors. Scanning electron microscopy imaging was used to image the morphology of the collagen barrier membranes loaded with the prolyl hydroxylase inhibitors dimethyloxalylglycine (DMOG) and L-mimosine (L-MIM) using the TM-1000 table top system (Hitachi, Tokyo, Japan). The samples were mounted on an aluminium stub and images were taken with backscattered electrons at 15 kV accelerating voltage at a 200-fold (The white bar represents 500 mm) magnification.

Table 1. Release of prolyl hydroxylase inhibitors from collagen barrier membranes (nmol/mg collagen barrier membrane).

DMOG L-MIM

hydroxylase inhibitors during the first few hours but do not impair the metabolic activity.

1h

3h

6h

24 h

48 h

Discussion

0.57 1.49

0.45 0.58

0.34 0.58

0.31 0.16

0.19 0.08

‘Hypoxia mimetic agents’ have been advocated as tools for oral surgery and periodontology as they can support wound healing and bone regeneration.9–11,15,18,19 Unclear, however, is how these molecules can be applied in clinically established strategies such as guided tissue regeneration. A new approach for a one-step procedure is to use devices such as collagen barrier membranes as carriers for prolyl hydroxylase inhibitors. Our study shows that prolyl hydroxylase inhibitors retain their biological activity when lyophilized onto collagen barrier membranes such as BioGideÕ . This is in line with studies showing that both CaSO4 and collagen sponges loaded with the prolyl hydroxylase inhibitor desferoxamine induce a pro-angiogenic response.14 Both, DMOG and L-MIM are rapidly released in the first 6 h from the carrier, as indicated by the increased VEGF production and reduction of proliferation in gingival and PDLFs in the monolayer cultures. Supernatants from collagen barrier membranes generated after 6 h showed a weaker cellular response. Although collagen barrier membranes were proposed as carriers for zoledronate and growth factors due to

Furthermore, these data suggest that a majority of the prolyl hydroxylase inhibitors are released during the first 6 h.

Prolyl hydroxylase inhibitors released from collagen barrier membranes within the first hours reduced proliferation Next we investigated the effect of the collagen barrier membrane preparations on proliferation (Figure 3) and metabolic activity (Figure 4) of the fibroblasts. Supernatants collected from DMOG and L-MIM loaded collagen barrier membranes reduced proliferation in fibroblasts from the periodontal ligament. A similar trend was observed in fibroblasts from the gingiva but did not reach the level of significance. Overall no significant effects were found on the conversion of MTT to formazan. These data support that collagen barrier membranes release the major amount of prolyl



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Collagen Barrier Membrane

Medium

Supernatant

Fibroblasts

VEGF

VEGF relative to the collagen barrier membrane control

(b) 7

DMOG L-MIM

6 5 4 3 2 1 0 1

3

6 Hours

24

48

VEGF

relative to the collagen barrier membrane control

(c) 7

DMOG L-MIM

6 5 4 3 2 1 0

1

3

6 Hours

24

48

Figure 2. Prolyl hydroxylase inhibitors released from collagen barrier membranes induce VEGF production in cell cultures within the first hours. (a) To reveal the release kinetic of prolyl hydroxylase inhibitors from collagen barrier membranes we utilized in vitro bioassay: Supernatants of collagen barrier membranes loaded with dimethyloxalylglycine (DMOG) and L-mimosine (L-MIM) were taken at hour 1, 3, 6, 24, and 48. The supernatants were subjected to cell cultures where their capacity to induce VEGF production was assessed with fibroblasts from the gingiva (b) and the periodontal ligament (c) using immunoassays for VEGF. The data points show the mean standard deviation relative to the untreated control. The dashed line represents the levels of the untreated control. The colour of the symbols represents the prolyl hydroxylase inhibitor. *p < 0.05, **p < 0.01.



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Medium

Supernatant

Fibroblasts

Proliferation

Proliferation relative to the collagen barrier membrane control

Proliferation relative to the collagen barrier membrane control

(b) 2.0

DMOG

1.8

L-MIM

1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 1

3

6 Hours

24

48

(c)

2.0

DMOG

1.8

L-MIM

1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 1

3

6 Hours

24

48

Figure 3. Prolyl hydroxylase inhibitors released from collagen barrier membranes within the first hours decrease proliferation in cell cultures. (a) The effect of supernatants from collagen barrier membranes supplemented with dimethyloxalylglycine (DMOG) and L-mimosine (L-MIM) of proliferation was assessed in cell cultures with fibroblasts from the gingival (b) and the periodontal ligament (c). Data points represent the mean standard deviation relative to the control collagen barrier membrane. The dashed line represents the levels of the untreated control. The colour of the symbols represents the prolyl hydroxylase inhibitor. *p < 0.05, **p < 0.01.

their net effects, studies assessing the release kinetic are limited to antibiotics and zoledronate.20–22 Our observations are in line with the burst release of zoledronate from 1 and 2 layered collagen barrier

membranes and data from prolyl hydroxylase, vitamins, and growth factors that were adsorbed onto bone substitute materials.20,23,24 However, the membranes release prolyl hydroxylase inhibitors within



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Medium

Supernatant

Fibroblasts

Metabolic activity

Metabolic activity relative to the collagen barrier membrane control

(b) 1.4

DMOG L-MIM

1.2 1.0 0.8 0.6 0.4 0.2 0.0 1

3

6 Hours

24

48

Metabolic activity relative to the collagen barrier membrane control

(c) 1.4

DMOG L-MIM

1.2 1.0 0.8 0.6 0.4 0.2 0.0 1

3

6 Hours

24

48

Figure 4. Effect of prolyl hydroxylase inhibitors released from collagen barrier membranes on the metabolic activity in cell cultures. (a) The effect of supernatants from collagen barrier membranes supplemented with dimethyloxalylglycine (DMOG) and L-mimosine (L-MIM) on the metabolic activity was assessed in cell cultures with fibroblasts from the gingiva (b) and the periodontal ligament (c). Data points represent the mean standard deviation relative to the control collagen barrier membrane. The dashed line represents the levels of the untreated control. The colour of the symbols represents the prolyl hydroxylase inhibitor.

the first 6 hours which is faster than found with vitamins and growth factors on bone substitute materials.23,24 However, this release kinetic is in line with the data from zoledronate from collagen barrier membranes where the highest release is found within the first 6 h.20

That only one dose of prolyl hydroxylase inhibitors was assessed is a clear limitation of this study. It remains unclear if higher doses of prolyl hydroxylase inhibitors might have prolonged the release and enhanced their pro-angiogenic effect or if this would have led to toxic responses. The dose used herein was



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based on a previous in vitro study with prolyl hydroxylase inhibitors in oral fibroblasts and data from bone substitute materials.15,19,25,26 The reduction of proliferation and metabolic activity in the first hour however suggest that higher concentrations are likely to have toxic effects. That we herein used DMOG and L-MIM which are not selectively inhibiting prolyl hydroxylases is another limitation as it might restrict their potential for clinical applications.15,19,27 Further studies are therefore indicated to find the optimal inhibitor and clinical dosage for oral tissue regeneration. BioGideÕ was used in the present study as a carrier for the prolyl hydroxylase inhibitors. This bi-layered collagen barrier membrane consists of type I collagen and type III collagen of porcine origin.7 The question arises whether other collagen barrier membranes might show a different release kinetic. Studies with the double-layered BioGideÕ and the single-layered collagen barrier membrane BME-10X composed of bovine collagen I showed no difference regarding the release of bisphosphonates.20 These data suggest that it is unlikely that monolayered collagen membranes show a different release kinetic. What we have contributed is to show that collagen barrier membranes supplemented with prolyl hydroxylase inhibitors can induce a pro-angiogenic response in fibroblasts from the periodontal ligament and the gingiva in vitro. The membranes release prolyl hydroxylase inhibitors in a burst like kinetic within the first hours. These findings provide the basis for preclinical studies to evaluate the regenerative capacity of prolyl hydroxylase inhibitors in periodontal surgery. Acknowledgements Collagen barrier membranes (BioGideÕ ) were provided by Geistlich Pharma AG (Wolhusen, Switzerland). The authors acknowledge Reinhard Gruber (Laboratory of Oral Cell Biology, School of Dental Medicine, University of Bern, Switzerland) for inspiration and thank Sophia Pilipchuk (Department of Biomedical Engineering, University of Michigan, USA) and Michael Edelmayer (Department of Conservative Dentistry and Periodontology, Medical University of Vienna, Austria) for proof-reading the manuscript. The authors thank William V Giannobile and his team (Department of Periodontics & Oral Medicine, School of Dentistry, University of Michigan) for their support and Christian Gruber and coworkers (Center for Physiology and Pharmacology, Medical University of Vienna) for help and advice with sample lyophilisation.

Declaration of conflicting interests None declared.

Funding This study was supported by grant 10-063 of the Osteology Foundation (Lucerne, Switzerland), and the Erwin Schro¨dinger Fellowship the Austrian Science Fund (FWF): J3379-B19.

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