Supplementary MaterialS1. Materials

Alizarin Red, pH4.1. (Sigma Aldrich #A5533 St. Louis, MO, USA) Angiopoietin-1, recombinant human (Ang-1, R&D Systems Inc. #923-AN-025, Boston, MA,

USA) Antibodies are listed in Table S1 L-Ascorbic Acid 2-phosphate (Vit. C, Fluka® Analytical #49752, Sigma-Aldrich, St.Louis,

MO, USA) Beta-glycerolphosphate (β-GP, Fluka® Analytical #50020, Sigma-Aldrich, St. Louis, MO, USA), Cerasorb®M (Curasan AG, Kleinostheim, Germany) Chicken egg, medium-sized from specified pathogen-free housing (Charles River

Laboratories, Wilmington, MA, USA). Chloramine T (Sigma-Aldrich #857319, St. Louis, MO, USA)) Collagenase (Worthington Biochem Corporation #LS005273, Lakewood, NJ, USA) Dexamethasone (Sigma Aldrich #D4902, St. Louis, MO, USA), Dimethyl Sulfoxide (DMSO, (Sigma Aldrich #D-841, St. Louis, MO, USA) Dulbecco’s Modified Eagle Medium - high glucose without pyruvate (DMEM, Invitrogen

#61965059, Thermo Fisher Scientific, Waltham, MA, USA) Dulbecco’s Phosphate Buffered Saline (DPBS, Gibco® #14190-094, Invitrogen, Thermo Fisher

Scientific, Waltham, MA, USA). Fetal Bovine Serum (FBS, Gibco® LOT# 41Q6208K, Thermo Fisher Scientific, Waltham, MA,

USA) Ficoll-Paque Plus ® (Amersham Biosciences PLC #17-1440-02, GE Healthcare, Little Chalfont,

Buckinghamshire, UK) bisBenzimide H33342 trihydrochloride (Hoechst33342, Sigma Aldrich #B2261, St. Louis, MO,

USA) 7-amino actinomycine D (7AAD, Sigma Aldrich #A9400, St. Louis, MO, USA) Hydrocortisone (HC, Sigma Aldrich #H0888-1G, St. Louis, MO, USA) 3-iso-butyl-1-methyl-xanthine (IBMX, Sigma Aldrich #I5879, St.Louis, MO, USA) Indo-methacine (IM, Sigma Aldrich #I7378, St. Louis, MO, USA), Iodine-125 (Perkin Elmer #NEZ033A005MC, Waltham, MA, USA) ITS+ premix: 100x, bovine insulin (10 µg/ml), transferrin (5.5 µl/ml), sodium selenite (5

µg/ml), linoleic acid (4.7 µg/ml, BSA (0.5mg/ml), (BD Biosciences #354352, Becton, Dickinson and Company Franklin Lakes, NJ, USA).

L-Aascorbateic acid -2-phosphate (AA, Vit.C, Fluka® Analytical #49752, Sigma-Aldrich, St.Louis, MO, USA)

L-Proline (Sigma Aldrich #P-0380, St. Louis, MO, USA) Minimal Essential Medium (MEM) + Glutamax™ (Gibco® #41090, Thermo Fisher Scientific,

Waltham, MA, USA) NanoSeed-18 (NS, seed crystal for CVD layers) and Liquid Diamond (LD, DP GAF 0-0.1) was

purchased from Microdiamant AG, Lengwil, Switzerland). Unprocessed Nanodiamond (ND) was purchased from Gansu Lingyun Corp. (Lanzhou, China)

Oil Red O (Sigma Aldrich #O0625-25G, St. Louis, MO, USA) Penicillin/Streptomycin, (Gibco #15140-122, Thermo Fisher Scientific, Waltham, MA, USA)

Secondary antibody: EnVision™ kit (Dako #K4008, Glostrup, Denmark) further contained goat anti-rabbit immunoglobulin, Proteinase K, protein block and AEC+.

Sephadex G-25 resin (GE Healthcare, #17-0851-01, Little Chalfont, Buckinghamshire, UK). Sodium pyruvate (Sigma Aldrich #P5280 St. Louis, MO, USA) Staurosporine (ST, Sigma Aldrich # S4400-.1MG, St. Louis, MO, USA)) TGFbeta1 (Chemicon #GF111, Merck Millipore, Billerica, MA, USA) Trypsin/EDTA (Gibco, #25300-05, Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) Vascular endothelial growth factor (VEGF, R&D Systems Inc., #293-VE-050, Boston, MA, USA)

Otherwise, all chemicals were purchased through VWR International GmbH

Table S1

Applied antibodies and Isotype controls

Antibody Isotype Company Clone ID Applied Concentration

CD 90 PE msIgG1,k BD 555596 5E10 0.5 µg

CD 105 PE msIgG2a Abcam ab69772 MEM-229 0.5 µg

CD146 PE msIgG2a BioLegend 342003 SHM-57 1 µg

CD44 PE msIgG1,k BD 550989 515 0.5 µg

CD73 PE msIgG1,k BioLegend 344003 AD2 0.5 µg

CD11b PE msIgG1,k BD 555388 ICFR44 0.5 µg

CD14 FITC msIgG2a Serotec MCA1568FT TÜK4 0.5 µg

CD31 PE msIgG1,k BD 555446 WM-59 0.125 µg

CD45 PE msIgG1,k BD 555483 HI30 0.25 µg

CD79a PE msIgG1,k ABIN302041 HM57 0.5 µg

CD19 PE msIgG1,k BioLegend 302207 HIB19 0.25 µg

vWF Polyclonal rabbit Abcam M0616 - 1:200

Isotype Company Concentration of stock Applied concnentration

FITC mouse IgG2a Biologend 400209 0.5 mg/ml 0.5 µg

PE mouse IgG1 Biolegend 400111 0.05 mg/ml 0.5 µg; 0.25 µg; 0.125 µg

PE mouse IgG2a Biolegend 400211 0.2 mg/ml 0.5 µg; 1 µg

S2-Supplementary Method 1:

Diamond material and nanoparticle analysis

Three nano-scaled diamond particles, different in size and derived by distinct fabrication procedures as disclosed in the manufacturer specifications were used in this study: Nanodiamond (ND), Nanoseed18 (NS) and Liquid Diamond (LD).

Commercial diamond samples:

NS and LD had been produced by shockwave synthesis. Liquid Diamond had a nominal crystallite size of 150 nm. NS produced by detonation synthesis exhibited a crystallite size of 4-8 nm agglomerated into 20-50 nm particle clusters.

Single-digit diamond particles:

Fully dispersed single-digit Nanodiamond (ND) was produced and size-selected as follows: Acid-purified detonation ND was beads-milled to a stable, colloidal aqueous solution as described previously (Krueger et al. 2005; Liang et al., 2009). The resulting material was filter-sterilized through a polyether sulfone membrane with an average pore size of 450 nm.

Particle dispersion

For sonication a Branson Ultrasonic-Homogenizer Sonifier II W-450, combined with a 5 mm microtip (max. 400 W, 19 850–20 050 Hz; Branson Ultrasonics, Emerson Electric Co., Danbury, CT, USA) was applied for 20 minutes.

Fourier-transform infrared (FTIR) spectroscopy:

Spectra were recorded using a Perkin-Elmer Spectrum One FTIR spectrometer (Perkin Elmer, Waltham, MA, USA) with a resolution of 4 cm-1. Nano-diamond powder (1 mg) was mixed with 100 mg KBr in an agate mortar. The mixture was pressed into a pellet under 10 tons load for 4 minutes, and the spectrum was recorded immediately thereafter. ND showed single crystallites of approximately 5-10 nm in diameter. For each spectrum, sixteen accumulative scans were collected. FTIR spectra with attenuated total reflectance were obtained with the aid of a Jasco FT/IR-410 (Jasco Corp., Japan).

Two major peaks at wave numbers of around 1630 and 3400 cm-1 representing OH groups were found in all samples. They originate from surface adsorbed water molecules but can be also derived from surface OH groups (free OH or from carboxylic groups). The sharp peak above 3600 cm-1 in the case of LD can be attributed to free OH groups. The signals slightly below 3000 cm-1 originate from C-H vibrations from hydrogenated surface atoms (methyl groups, methylene bridges). Furthermore, the samples showed peaks for carbonyl groups at ~1700 cm-1 and ether groups (around 1000-1300 cm-1). These were likely due to the oxidative purification of the samples using strong mineral acids.

Particle analysis

For light microscopic analysis the individual nDPs were diluted in PBS at pH7.4. The suspensions were dispersed for 15 min as described above and imaged under phase contrast transillumination using a Zeiss Axiophot photomicroscope equipped with a digital camera.

Zeta potentials (ZP) were examined over a wider pH range in particular to assess dispersion stability at physiological pH. Dispersion stability was expected above +30 mV or below -30 mV. ZP was recorded between pH 3 and 9 with a Malvern-Zetasizer Nano-ZS (Malvern Instruments, Worcestershire, UK). For particle size and zeta potential measurements, diamond particles were suspended in 1 mL of water and injected into a folded capillary cell. The values of the particle sizes correspond to the weight converted distribution. Particle size and ZP were measured without prior centrifugation, taking all particles into account.

Thermo-gravimetric analysis (TGA) was carried out at a heating rate of 10 K min-1 with the aid of a Perkin Elmer STA 6000 (Perkin Elmer, Waltham, MA, USA) carried through the.

A Vario Micro Cube (Elementar Analysesysteme GmbH, Hanau, Germany) was employed for elemental analysis.

Scanning electron microscopy (SEM) was performed with the aid of an InLens detector equipped Zeiss Ultra Plus SEM (Zeiss GmbH, Oberkochen, Germany) applying acceleration voltages of 0.5 (Fig 1 E, F) or 1V (Fig 1D). For sample preparation a drop of diamond dispersion was spin-coated at 2000 rpm for 1 minute onto a Si wafer (orientation <111>, Plano GmbH, Germany) as a substrate.

References:

Krueger, M. Ozawa, F. Kataoka, T. Fujino, Y. Suzuki, A. E. Aleksenskii, A. Y. Vul'E. Osawa, Carbon 2005, 43, 1722.

Liang Y, Ozawa M, Krueger A. A general procedure to functionalize agglomerating nanoparticles demonstrated on nanodiamond ACS Nano. 2009 3:2288-96.

S2: Supplementary Material & Method 2:

For in vitro biological testing of pristine nDP, we employed primary human mesenchymal stromal cells (MSC), isolated from healthy individuals. They exhibit high proliferation potential in culture, multipotential differentiation capacity in vitro as well as immunosuppressive activity (Dazzi, et al 2012). MSC were retrieved from cancellous bone of iliac crest biopsies of systemically healthy individuals who had undergone reconstructive surgery (Klepsch et al., 2013).

Cultivation and Treatment of human bone derived mesenchymal stromal cells (MSC)

Isolation

A small biopsy of substantia spongiosa osseum, which otherwise would have been discarded based on necessary bone molding/recontouring prior to insertion into the recipient site was taken to further investigation under an Institutional Review Board-approved protocol after having obtained patients’ written consent, written parental consent or legal guardian consent. After surgery, the bone was transferred into Minimal Essential Medium (MEM) + Glutamax™ -I supplemented with 16.6%, 100 units/ml penicillin, 100 µg/ml streptomycin for transportation from the operating room to the clean room at RT. The biopsies were fragmented and marrow cells were isolated from pieces (20-100 mm3) by treatment with collagenase (2.5 mg/ml in MEM) for 2-4 hours at 37°C, 20% O 2, 5% CO2 depending on tissue sample size. Thereafter, the specimen was centrifuged (400 x g, 3 minutes). Flow-through (total bone marrow cells) were resuspended in 4 ml fresh medium, carefully loaded on 5 ml Ficoll-Paque Plus® gradient using a sterile 15-ml tube and centrifuged at 2,500 x g for 30 minutes without breaks. Mononuclear cells were harvested from the interphase (density <1.075 g/ml), collected by centrifugation (1,500 x g, 15 minutes) and washed once in warm medium (resuspension in 5 ml and centrifuged (400 x g, 3 minutes). After counting, cells were seeded with a density of 200,000 cm-2. They were cultured in a tissue culture dish at a density of 0.2 – 0.5 x 106 cells cm-2 either at 21% O2, 5% CO2, 37°C (Heraeus®, HERACell™ 240 Thermo Fisher Scientific, Waltham, USA) or at 3% O2, 5% CO2, 37°C (Forma™ Series II 3110 Water-Jacketed CO2 Incubator, Thermo Fisher Scientific, Waltham, USA). After 24 hours, the non-adherent cell fraction was removed by washing twice with warm, sterile DPBS. After primary culture reached approximately 30-50 % confluence, cells were first washed twice with warm and sterile DPBS, and subsequently treated with 0.05% trypsin / 1 mM EDTA for 3-5 minutes at 37°C. Cells were harvested, washed once in MEM and further expanded at a density of 50 cells cm-² or gently frozen in freezing medium (MEM supplemented with 100 units/ml penicillin, 100 µg/ml streptomycin, 30% FCS, 0.5% DMSO to a final density of 1 x 10 6

cells/ml, -70°C with the aid of Cryo Freezing Container (Nalgene, Mr. FrostyTM, Thermo Fisher Scientific, Waltham, MA USA) and, 2-3 days later, transferred to liquid nitrogen.

Culture

Cells from primary cultures were passaged (see above) and reseeded at a density of 50 cells cm-2 to select for MSC.

Figure S2.1: Primary culture of freshly isolated human iliac crest derived mesenchymal stromal cells (MSC), adhering to a plastic petri dish.

Characterization of MSC properties

Flow cytometric analysis: cells were harvested by 0.05% trypsin/EDTA treatment, then washed and suspended in sterile DPBS. Aliquots of 50 µl (0.2–1x106 cells) were incubated with indicated antibodies or isotype controls (Table 1) in the dark at 4°C for 30 minutes and then washed with sterile DPBS. Surface marker expression was analyzed with the aid of a FACS Canto flow cytometer and FACS-DivaTM Software (BD PharmingenTM, BD Biosciences, Franklin Lakes, NJ, USA).

Figure S2.2: Phenotyping of cultivated human bone derived mesenchymal stromal cells (MSC). Flow cytometric analysis of MSC surface markers. Histograms of present (top row) and absent (bottom row) surface markers (black lines) are shown together with the respective isotype control (in red).

In vitro differentiation: MSC were plated at 50 cells cm-2, grown to 80-90% confluence prior differentiation and transfer to ambient atmosphere, 3% O2, 5% CO2, 37°C (Forma™ Series II 3110 Water-Jacketed CO2 Incubator, Thermo Fisher Scientific, Waltham, USA).In course of starting the differentiation process, cultures were transferred to an atmosphere of 21% O2, 5% CO2, 37°C (Heraeus®, HERACell™ 240 Thermo Fisher Scientific, Waltham, USA)Osteogenesis was stimulated using growth medium containing 1 mM beta-glycerolphosphate, 100 nM dexamethasone, and 0.5 mM ascorbate-2-phosphate. Induction medium was changed twice per week for a period of 3 weeks. For evaluation, the cells were fixed with 4% para-formaldehyde/phosphate- buffered saline (PBS) for 10 minutes. Cells were washed twice with PBS pH4.1 and stained for 20 minutes with Alizarin Red pH4.1. For adipogenic differentiation of MSC, growth medium was supplemented with 1 mM dexamethasone, 60 mM indo-methacine (IM), 0.5 mM 3-iso-butyl-1-methyl-xanthine (IBMX), and 0.5 mM hydrocortisone (HC) for three weeks. After fixation with 4% para-formaldehyde/phosphate- buffered saline (PBS) for 10 minutes, lipid vesicles in differentiated cells were stained with 0.7% Oil Red O in 85% propylene glycol for 20 minutes.

To differentiate MSC towards the chondrogenic lineage, 0.5x106 MSC were seeded in a 15-ml tube and cultured in DMEM high glucose without pyruvate as a pellet culture. Medium was supplemented with 0.2 mM L-Ascorbic Acid 2-phosphate, 1 mM sodium pyruvate, 10 ng/ml TGFbeta1, 100 nM dexamethasone, 40µg/ml L-Proline and ITS+ premix (100x, bovine insulin (10 µg/ml), transferrin (5.5 µl/ml), sodium selenite (5 µg/ml), linoleic acid (4.7 µg/ml, BSA (0.5mg/ml)).

Figure S2.2: Standardized tri-lineage differentiation of human bone marrow derived mesenchymal stromal cells. A) Successful adipogenic differentiation is indicated via lipid vacuoles in the cytoplasm, stained with Oil Red O. B) Alzarin Red stains calcified deposits by osteogenic differentiated MSC. C) A histological specimen of a chondrogenic differentiated MSC pellet (3D culture) stained immunhohistochemically for collagen II.

500 µm 300 µm CBA50 µm

nDP application

nDP suspensions were sterilized using gamma irradiation (25kGy) and deagglomerated using a Branson Ultrasonic-Homogenizer Sonifier II W-450, combined with a 5 mm microtip (max. 400 W, 19 850–20 050 Hz; USA) for 20 minutes prior to application. 0.02, 0.2, 1 or 2 mg ND, NS or LD (all dispersed in water) per ml medium were added directly into the culture dish, gently dispersed and incubated for 24 or 72 hours. Control cultures were free of nDP or treated with 0.4 µM staurosporine to induce apoptosis, 24 hours prior to the end of the experiment.

Cell death

All wells were washed twice with 37°C warm Dulbecco’s phosphate buffered saline (DPBS). Wells containing high amounts of nDP samples were washed extremely carefully until the cells were clearly visible. 1 µl/ml of fluorescent dyes Hoechst33342 and 7-amino actinomycine D (7AAD were added and incubated for 15 minutes at 37°C, 5% CO2 and 3% O2

in a dark environment. Thereafter, supernatant was removed and the cells were rinsed with DPBS. Prior to analysis, 500 µl DPBS were added to each well. The dyes were excited with UV-light. Per well, at least three fields of view were analyzed and the number of cells exhibiting nuclear 7-AAD fluorescence was accounted.

References

Dazzi, F. Lopes, L.; Weng, L. Mesenchymal stromal cells: a key player in innate tolerance?, Immunology. 2012, 137:206-213.

Fehrer C, Brunauer R, Laschober G, Unterluggauer H, Reitinger S, Kloss F, Gülly C, Gassner R, Lepperdinger G. Reduced oxygen tension attenuates differentiation capacity of human mesenchymal stem cells and prolongs their lifespan. Aging Cell 2007 6:745-57.

Klepsch, S. Jamnig A, Trimmel D, Schimke M, Kapferer W, Brunauer R, Singh S, Reitinger S, Lepperdinger G. Isolation of mesenchymal stem cells from human bone and long-term cultivation under physiologic oxygen conditions. Methods Mol Biol. 2013;976:99-109. doi: 10.1007/978-1-62703-317-6_8

S3: Supplementary Method 3:

Radiolabelling of bioactive factors

Recombinant human angiopoietin-1 (Ang-1, R&D Systems Inc., 923-AN-025, Boston, MA, USA) and Vascular Endothelial Growth Factor (VEGF, R&D Systems Inc., 293-VE-050, Boston, MA, USA) were labeled with iodine-125 (Perkin Elmer, NEZ033A005MC, Waltham, USA). 1 µg of VEGF or Ang-1 were dissolved in 10 µl PBS, and 100 µCi iodine-125 together with 20 µl 2% chloramine T were added, and thoroughly mixed. After 5 minutes, unreacted iodine and the chloramine T were separated from the protein using a disposable PD-10 desalting column packed with Sephadex G-25 resin (GE Healthcare, 17-0851-01, Little Chalfont, Buckinghamshire,UK). The labeled protein was stored at 4°C. Approximately 1.3 radioisotopes could be incorporated per protein. Before use, the specific activity of the radiolabeled protein was diluted ten-fold by addition of cold, unlabeled protein.

Figure S3: Experimental procedure to measure physisorption of radio-labelled proteins to nDP surfaces. Iodininated proteins dissolved in PBS were functionalized to nDP surfaces for 24h at 4°C on a rotator. After centrifugation, supernatant was taken off and radiation was measured. The pellet was resuspended in PBS and shear forces applied (W1). nDP were again centrifuged and the process was repeated three times (W1-3).

Method adopted from Bailey, The chloraminutese T Method for radiolabeling Protein. Protein Protocols Handbook 2002 963-965 from: http://link.springer.com/protocol/10.1385%2F1-59259-169-8%3A963

S4: Supplementary Method 4:

The specific biological activities of the selected growth factors after immobilization to nDP have been assessed. For this purpose we used the Hen’s Egg Test on Chorioallantoic Membrane (HET-CAM), which has previously been introduced for pristine and biofunctionalized nDP testing (Wierzbicki et al. 2013a; Wierzbicki et al 2013b).

The developing chicken has an extended vessel system which engulfs the yolk thereby serving as a respiratory, excretory and storage organ for the embryo. As previously shown, this emerging biological complexity is highly suitable to assess the activity of vessel inductive factors bound to synthetic carriers. In parallel, also potential hazards of the applied nDP can be easily identified by scrutinizing CAM and vessel integrity, and pattern formation of the vascular plexus. Further on, the hen’s egg is readily available, cost-efficient and relatively easy to manipulate, why it is a suitable test object for the here presented studies. Besides photomicrographs, which were taken from the wider surroundings of the graft, in addition also histological analysis was undertaken. The vessels were visualized by immunostaining and both the number and the lumen diameters were accounted and results were statistically validated.

Hen’s Egg Test on Chorioallantoic Membrane - HET CAM

Before commencing the in ovo breeding, the egg shell was carefully cleansed with 70% EtOH and placed in a breeder (Brut Control by Heka, Germany) at 36.8°C, 55% humidity revolving the eggs for 180° every 30 min.

After breeding the eggs until the corresponding embryonic development day (EDD) 3, the rotation was stalled. After an hour, the eggs were cleaned with 70% EtOH and the upper side was marked, as the embryo is located there. The eggs were carefully opened starting from the opposing side to avoid impact on the embryo. The entire egg content (white and yolk together with the embryo) was displaced from the shell into UV and 70% EtOH sterilized 10x10cm weighing dishes under sterile conditions and avoidance of any damage to yolk and vasculature. Viable embryos have an approximate size of 1 mm surrounded by a vascularized disc of approximately 1-2 cm in diameter. They were covered and further incubated for 7 days at 36.8°C and 85% humidity. An untreated embryo at EDD 12 is shown in Figure S3.1.

Application of nDP

We exposed the nDP as an onplant to the CAM. This application method was chosen, as it ensured experimental viability, a high grade of reproducibility and ease of handling.Per onplant, 0.6 mg nDP bearing 20 ng of protein, suspended in 30 µl DPBS, were added to 30 µl rat tail collagen type I (3.66mg/ml (BD Biosciences, #354236) matrix with 0.2N NaOH and 10X Gibco® DMEM (Gibco #52100, 0.2L, Thermo Fisher Scientific, Waltham, MA, USA) were mixed in a ratio of 8:1:1.

A total volume of 60 µl per graft (4-5 grafts per CAM) were spotted onto sterilized Parafilm® prior to application and incubated for 45 minutes at 37°C to promote and control solidification of the nDP collagen mixture. Subsequently, the polymerized grafts were transferred to the CAM of 10-days old embryos.

The chicken embryo was then incubated for another 4 days. Images of the CAM bearing collagen-containing nanoparticles were acquired using a stereomicroscope equipped with a Nikkon DSLR camera.

Figure S3.1: Image of a 12 day old chicken embryo removed from the egg shell

Histological analysis

The onplants with the juxtaposed membranes were explanted, paraffin embedded and sectioned for subsequent immunohistochemical evaluation with an antibody against von Willebrand Factor to label vasculature. Diameters and numbers of vessels were thoroughly measured and evaluated.

In detail, the dissected specimens were rinsed in PBS, fixed overnight in 4% paraformaldehyde and briefly counterstained with toluidine blue. Paraffin (Paraplast, Carl Roth&Co. GmbH, #X.880.1, Karlsruhe Germany) was molten at 57°C. The specimen were dehydrated in 70% EtOH for 2h, in 80% EtOH twice for 30 minutes, in 90% EtOH twice for 20 minutes, twice in 100% EtOH for 20 minutes, and lastly transferred into methylbenzoate (Sigma Aldrich, #M2, 990-8 St. Louis, MO, USA) for about 15 minutes followed by two consecutive 10-minutes incubation steps with chloroform. The specimens were incubated in paraffin at 55-57°C overnight. The objects were transferred two times every 2 hours into fresh paraffin, at 55-57°C. After embedding, the ice cold block was trimmed and 4-8 µm sections were cut using a rotary microtome (Shanton Finesse E+ by Thermo Electro Cooperation). The sections were deparaffinized with xylol for 5 minutes twice, 100%, 96% and 70% EtOH for 2 minutes twice, followed by washing with deionized water for 2 minutes. The sections were treated with the EnVision™ kit (Dako #K4008, Glostrup, Denmark) according to the manufacturer’s instruction applying Proteinase K (Dako #S3020, Glostrup,

Denmark) for 15 minutes at 37°C. Thereafter, samples were washed with PBS for 5 minutes and further incubated with 0.03% hydrogen peroxide for 30 minutes followed by PBS for 5 minutes, protein block (Dako, X0909, Glostrup, Denmark) for 20 minutes, and anti-von-Willebrand factor (vWF) (human, raised in rabbit, Abcam, ab6994, UK) diluted at 1:200 with antibody diluent for 90 minutes at room temperature. The slides were washed with PBS pH7 and then incubated with horse radish peroxidase (HRP) –labeled goat anti-rabbit immunoglobulin (Dako EnVision™ K4008, Glostrup, Denmark) for 30 minutes. The specimen were washed with PBS for 5 minutes and treated with 3-amino-9-ethyl-carbazole (AEC+, Dako EnVision™ #K4008, Glostrup, Denmark) for 15 minutes. Solutions were washed off with deionized water for 5 minutes. The specimen were counterstained for 30 sec using Mayer’s Hemalum solution followed by 5 minutes washing in tap water and deionized water. Slides were coversliped using Aquatex® (Merck Millipore, #108562, Billerica, MA, USA) as a mounting medium.

References

Lüpke, N.P. (1986) HET (Hen's Egg Test) in Toxicological Research. In: Skin Models. Models to Study Function and Disease of Skin(eds. Marks, R. & Plewig, G.). Published by Springer-Verlag, pp 282-291.

INVITTOX Protocol Nr. 47 (1990), ISSN #0960-2194

Spielmann, H., Gerner, I., Kalweit, S., Moog, R.,Wirnsberger, T., Krauser, K., Kreiling, R., Kreuzer,H., Lupke, N.P. Miltenburger, H.G.Muller, N. Murmann, P. Pape, W. Siegmund, B. Spengler, J. Steiling, W. & Wiebel, F.J.(1991) Interlaboratory assessment of alternatives to the Draize eye irritation test in Germany. Toxic. in Vitro, 5 No.5/6, 539-542.

Wierzbicki, M.; Sawosz, E.; Grodzik, M., et al. Carbon nanoparticles downregulate expression of basic fibroblast growth factor in the heart during embryogenesis, Int J Nanomedicine. 2013, 8, 3427-3435.

Wierzbicki, M.; Sawosz, E.; Grodzik, M.; Prasek, M.; Jaworski, S.; Chwalibog, A. Comparison of anti-angiogenic properties of pristine carbon nanoparticles, Nanoscale Res Lett. 2013, 8, 195.

S5: Supplemental Material 5:

Ang-1 induces angiogenesis hence forming new arteries and veins from preexisting vessels (Fagiani et al. 2013). We therefore investigated Ang-1 physisorbed to CS via nDP interaction. Cerasorb®M (CS) varnished with nDP permitted the immobilization of minute amounts of Ang-1 to the scaffold. The input of physiologic concentrations not only warrants an appropriate biological response yet shall greatly preclude unwanted adverse ectopic effects. Moreover, application of the factor only shortly before implantation at the site of operation jacks up the chances that protein activity is preserved.

In vivo application of ND functionalized with Ang-1

Varnishing of scaffold with nanoparticles

Cerasorb®M (CS) a β-tri-calcium phosphate (Ca3(PO4)2) free from phase shift exhibited pore sizes of 5-500 µm and a density of 3,081 g/cm3 was provided by (Curasan AG, Kleinostheim Germany). Cylinders (~1cm3) were perfused with 1.5 ml of 2% (w/v) ND with 1.5 bar pressure and a flow rate of 10 ml min-1 for 5 minutes. Vacuum desiccation ensured uniform distribution of ND within the scaffolds, and the retained amount was measured as described Hartl et al. 2004. The surface area was unchanged as assessed by mercury intrusion porosimetry. The modified scaffold was drained with 1 ml Ang-1 (1 µg ml-1 in PBS). As a control, scaffolds were varnished with ND bearing covalently bound Ang-1.

Sheep surgerySheep surgery was performed as described previously (Kloss et al., 2008). Prior to animal experimentation the specific design and conduct was approved by the Austrian Ministry of Science (permission BMWF-66.011/0146-II/3b/2010).

Healthy six-year-old female sheep weighing 75 ±5kg were housed in groups of 5. The animals were fed three times a day with free access to water. Prior to surgery they were kept singly, fasted overnight while having free access to water. Animals were frequently visited (Figure S5.1 A).

20 minutes before operation, the individual animal received an injection of 1 mg atropin sulfat (Nycomed, Takeda Pharma GmbH, Konstanz, Germany). Subsequently an indwelling permanent venous catheter was placed in the ear vein. General anesthesia was obtained by intravenous application of 15 mg kg-1 Fentanyl (Janssen-Cilag GmbH, Neuss, Germany). The animal was placed onto a warming pad at the operating table. After intubation (Fig. S5.1 B), controlled ventilation was carried out (Vt approximately 800 ml, f=18) and 1-2 vol% isofluran (Baxter AG, Volketswil, Switzerland) was administered with the aid of a flow-through vaporizer (Figure S5.1 B). Respiration was continuously monitored and adjusted. To prevent exhalation, a probe was placed into the rumen.

After shaving the head, the skin was desinfected by repeated washing with antiseptics (Skinsept, Ecolab, Monheim, Germany) and the disinfected area was covered with sterile tissues (Figure S5.1 C). A sagittal incision was performed from the forehead to the occipital bone and after coagulation the skull was exposed by subperiostal dissection. The positions of drill holes were specified and marked (Figure S5.1 D). 10 critical-size-defects (1 cm diameter) with full penetration without lacerating the dura mater were created in each animal with a trephine burr (Figure S5.1 E). CS cylinders were implanted into 1-cm drill holes in the calvaria

(Figure S5.1 F). Defects were randomly filled with pure Cerasorb®M (clinical standard, as control), Cerasorb®M with Nanodiamond and covalently bound Ang-1 (CS+ND+covAng1) as a positive control, or Cerasorb®M with Nanodiamond and physisorbed Ang-1 (CS+ND+phyAng-1). Protruding ends of CS were carefully removed to preserve the texture of the calvaria. Monocortical titanium screws were inserted to ease identification of CS positionings after sacrification. The periosteum provides vascularity and plays an essential role in the bone healing and remodeling processes, Therefore the periosteum adjacent and on top of the defects was carefully removed before suturing. Thus, CS was solely touched by the epicranius muscle layer. Subcutaneous sutures and cutaneous sutures were done for wound closure.

During recovery phase the following analgesic regimen was applied: 5-10 μg kg-1

Buprenorphin (Reckitt Benckiser Pharmaceuticals, Inc., Richmond, VI, USA) intravenously every 6 hours, 2 mg kg-1 Flunixin (Serumwerk BernburgAG, Bernburg, Germany) subcutaneously every 24 hours and at the second day post operation Fentanylpflaster (1APharma GmbH, Oberhaching, Germany) 100 μg h-1 lasting 72 hours.

One and three months after implantation, animals were sacrificed. Before, throughout and after surgery, the animals were attended accordingly. Anesthesia, analgesia and potential antibiosis were conducted by qualified personnel.

Figure S5.1. Sheep surgery: (A) Shortly before operation, sheep were delivered from the animal facility to the animal experimental laboratory; (B) After primary anesthesia, sheep were intubated for controlled ventilation and isofluran inhalation; (C) Head was shaved and skin disinfected; (D) incision of the skin and positioning of drill holes; (E) fully penetrated bone defects; (F) holes filled with Cerasorb M cylinders

Immunohistochemical analysis

Bone was explanted, fixed with 4% phosphate-buffered paraformaldehyde (PFA) and embedded in Technovit® 9100NEW (Hearaeus Kulzer #64715444, Hanau, Germany) following the manufacturer’s instructions (Kloss et al, 2013) to perform hard-tissue sections.

After embedding, the hardened blocks were cut in the middle (longitudinal direction), one half was subjected to histology staining and the other half was used for immune-histochemical analysis. (Donath et al., 1982). Sections of 10-µm thickness were used for immunohistochemical staining according to previous reported protocols with minor modifications (Rippstein et al., 2006). The samples were incubated with the mouse-anti-von-Willebrand Factor (vWF) (Dako, Denmark, 1:200 dilution) overnight at 4°C. After washing with Tris-buffered saline (TBS), samples were incubated with peroxidase-labelled secondary antibody (EnVision+TM Dual Link System-HRP, Dako, Glostrup, Denmark) for 30 minutes. at RT. Peroxidase activities were visualized with 3-amino-9-ethylcarbazole (AEC) substrate chromogen system (Dako, Glostrup, Denmark). The samples were mounted with aqueous mounting medium (Aquatex®, Merck, Germany) for observations.

Histomorphometry

Semi-quantitative measurements of von Willebrand factor stained sections were performed with Nikon NIS-elements software. The image of the whole sample was taken with Nikon Eclipse 80i microscope at 100x magnification coupled with a CCD camera using the stitching function from NIS-element software. The region of interest was defined as the area where defect site containing with tissue/scaffold reaching from periphery of the host bone. Vessels were identified as luminal structures containing erythrocytes and counted manually in a blinded manner by two independent observers. The vessel density was quantified using the number of positively stained vessels divided by the total area of interest, which was then presented as number of vessels per area (vessels mm-2). All measurements were performed with the open source software Fiji (www.Fiji.sc) using the “Cell Counter” plugin. Statistics were calculated via Student’s T-Test and one-way ANOVA.

References

Donath K, Breuner G. A method for the study of undecalcified bones and teeth with attached soft tissues. The Sage Schliff (sawing and grinding) technique. J Oral Pathol. 1982;11:318

Fagiani, E.; Christofori, G. Angiopoietins in angiogenesis, Cancer Lett. 2013, 328, 18-26.

Hartl A, Schmich E, Garrido JA, Hernando J, Catharino SC, Walter S, et al. Protein-modified nanocrystalline diamond thin films for biosensor applications. Nat Mater. 2004;3:736-42

Kloss, F. R et al. The role of oxygen termination of nanocrystalline diamond on immobilisation of BMP-2 and subsequent bone formation, Biomaterials. 2008, 29, 2433-2442. References

Kloss, F. R.; Singh, S.; Hachl, O., et al. BMP-2 immobilized on nanocrystalline diamond-coated titanium screws; demonstration of osteoinductive properties in irradiated bone, Head Neck. 2013, 35, 235-241.

Suliman, S. et al. Release and bioactivity of bone morphogenetic protein-2 are affected by scaffold binding techniques in vitro and in vivo, J Control Release. 2015, 197, 148-157.

S6. Supplementary Method 6

Statistics

Experimental values were displayed as arithmetic means with respective standard deviations. For statistical validation in which experimental results were validated pairwise, the two-tailed Student’s t-test was performed. In other cases one-way ANOVA was carried out.

S7: Supplementary Table S2: Compilation of nDP characteristics: For evaluation purposes the examined properties of the individual particles were compiled. Biocompatibility of very small ND and medium-sized NS was superior over the rather large LD. Ang-1 physisorbed well onto all nDP, yet only readily dissociated from NS. VEGF attached best to LD. When bound to ND, VEGF exhibited the steadiest interaction, while becoming readily available when physisorbed to NS and LD. Ang-1 and VEGF remained bioactive only when associated with ND and NS. Ang-1 performed superior when bound to NS, VEGF scored best when combined with ND.

Nanodiamond NanoSeed Liquid DiamondSizingZeta potential (mV at pH 7.4) 34 29 7Size (nm) 7 ±5 30 ±5 90 ±10Toxicity%Cell viability * 24 hours

72 hours95.8598.22

95.9398.66

96.8898.72

%Cell metabolic activity * 24 hours 72 hours

93.7283.96

87.8684.28

92.4170.73

Biocompatibility + + ±Protein bindingAng-1: association † 45.61 48.90 52.38Release of potentially active Ang-1 ‡ 13.80 25.24 15.53Ang-1 association|dissociation + ± + ++ + ±VEGF: association † 28.85 23.36 36.22Release of potentially active VEGF ‡ 10.63 14.43 21.38VEGF association|dissociation + ± ± + + ++In vivo assessment (HET-CAM)Pristine nDP: plexus remodeling n.s. § n.s. § n.s. §

Bioactive Ang-1: vessel induction (fold change) ||

2.13 ¶ 2.22 ¶ 0.67

Ang-1: fold change in medium and large lumen dimensions #

3.29 5.29 1.61

nDP-Ang-1 bioactivity|lumen dimension + + + ++ - ±Bioactive VEGF:

vessel induction (fold change) ||3.82 ¶ 2.04 ¶ 1.06

VEGF: fold change in medium and large lumen dimensions #

4.26 4.09 3.24

nDP-VEGF bioactivity|lumen dimension ++ + + + - +

*) Human bone derived mesenchymal stromal cells treated with 0.2 mg ml-1 nDP†) % bound after physisorption of 50 ng to 0.2 mg nDP‡) Release from nDP after three washes: % yield of initially provided for binding§) Cross-comparisons between collagen onplants loaded with pristine nDP, also in reference to empty collagen onplants yielded non-significant differences (n.s.) as validated by ANOVA ||) Comparison of onplants loaded with pristine and functionalized nDP

¶) Significantly different as validated by Student’s T-test#) Comparison between empty collagen onplants and those loaded with functionalized nDP