Name: biological compatibility profile on biomaterials for bone regeneration
Uploaded: 2018-11-16T13:00:00.000+00:00
Duration: 10 min 28 s
Description: Watch this Scientific Journal Video about Biological Compatibility Profile on Biomaterials for Bone Regeneration at JoVE.com

This research project has received funding from the European Union&#39;s Seventh Framework Programme (FP7/2007-2013) under grant agreement no. 263363.

<ol>
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V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+roles+of+immune+cells+in+bone+healing;+what+we+know,+do+not+know+and+future+perspectives.">The roles of immune cells in bone healing; what we know, do not know and future perspectives.</a> Injury. 47 (11), 2399-2406 (2016).</li><li>Changi, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biocompatibility+and+immunogenicity+of+elastin-like+recombinamer+biomaterials+in+mouse+models.">Biocompatibility and immunogenicity of elastin-like recombinamer biomaterials in mouse models.</a> Journal of Biomedical Materials Research. Part A. 106 (4), 924-934 (2018).</li><li>Laczko, J., Levai, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+differential+staining+method+for+semi-thin+sections+of+ossifying+cartilage+and+bone+tissues+embedded+in+epoxy+resin.">A simple differential staining method for semi-thin sections of ossifying cartilage and bone tissues embedded in epoxy resin.</a> Mikroskopie. 31 (1-2), 1-4 (1975).</li><li>Nakayama, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polarized+osteoclasts+put+marks+of+tartrate-resistant+acid+phosphatase+on+dentin+slices--a+simple+method+for+identifying+polarized+osteoclasts.">Polarized osteoclasts put marks of tartrate-resistant acid phosphatase on dentin slices--a simple method for identifying polarized osteoclasts.</a> Bone. 49 (6), 1331-1339 (2011).</li><li>Deguchi, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+model+of+bone+to+facilitate+measurement+of+adhesion+forces+and+super-resolution+imaging+of+osteoclasts.">In vitro model of bone to facilitate measurement of adhesion forces and super-resolution imaging of osteoclasts.</a> Scientific Reports. 6, 22585 (2016).</li><li>Epstein, N. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+analysis+of+noninstrumented+posterolateral+lumbar+fusions+performed+in+predominantly+geriatric+patients+using+lamina+autograft+and+beta+tricalcium+phosphate.">An analysis of noninstrumented posterolateral lumbar fusions performed in predominantly geriatric patients using lamina autograft and beta tricalcium phosphate.</a> Spine Journal. 8 (6), 882-887 (2008).</li><li>Epstein, N. 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D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repair+of+a+critical-sized+calvarial+defect+model+using+adipose-derived+stromal+cells+harvested+from+lipoaspirate.">Repair of a critical-sized calvarial defect model using adipose-derived stromal cells harvested from lipoaspirate.</a> Jove-Journal of Visualized Experiments. (68), (2012).</li><li>Gosain, A. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteogenesis+in+calvarial+defects:+contribution+of+the+dura,+the+pericranium,+and+the+surrounding+bone+in+adult+versus+infant+animals.">Osteogenesis in calvarial defects: contribution of the dura, the pericranium, and the surrounding bone in adult versus infant animals.</a> Plastic and Reconstructive Surgery. 112 (2), 515-527 (2003).</li><li>Levi, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dura+mater+stimulates+human+adipose-derived+stromal+cells+to+undergo+bone+formation+in+mouse+calvarial+defects.">Dura mater stimulates human adipose-derived stromal cells to undergo bone formation in mouse calvarial defects.</a> Stem Cells. 29 (8), 1241-1255 (2011).</li><li>Wang, J., Glimcher, M. 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The authors have nothing to disclose.

Here, we show a multidisciplinary approach for the preclinical assessment of biocompatibility for representative biomaterials developed for bone regeneration and repair. We tested the responses of OBs, OCs, and the in vivo healing response in a critical bone defect model in mice as well as in vitro and in vivo immune responses. We aimed to demonstrate how the assays work and summarize the data and conclusions derived from the examination of the biomaterials. We show that our strategy generates ...

Bone repair is a complex process that begins with hematoma formation, inflammation, callus formation and then remodeling1,2. However, bone regeneration potential is limited to the size of the bone fracture1,3. For instance, large bone fractures caused by trauma, cancer or osteoporosis may not heal and are termed non-union bone fractures. These bone lesions often require treatment to promote healthy physiological bone repair and regeneration. Currently, autograft and allograft bone transplantation is the approach of choice4 with 2.2 million bone replacement procedures annually5. Though these procedures have a high success rate, there may be complications, for example, limited availability of bone, infection, donor site morbidity, and rejection4. New alternatives for bone tissue engineering are being sought to address these challenges.
The design of biomaterials based on natural or synthetic polymers, bioceramics or metals in combination with cells and bioactive molecules is on the rise6. Our current understanding of physiological bone healing and healing in the context of biomaterials depends on multiple factors such as mechanical properties and multiple local and systemic factors including cells from the circulation and fracture site7,8,9. Biomaterials for bone regeneration aim to promote osteogenicity and osseointegration10 and are ideally biocompatible, biodegradable, and porous (promoting cell migration, oxygen, and nutrients). They also need to be sufficiently strong to support the fracture site to relieve pain. Additionally, inflammatory factors are required to initiate the healing process. However, if the biomaterial induces excessive inflammation and allergic responses, this might limit or inhibit bone healing11,12. Thus, an interdisciplinary approach is necessary to evaluate biomaterials developed for bone repair.
In this study, we present a pre-clinical evaluation of representative materials, 1) Orthovita Vitoss foam which is a commercially available cancellous bone graft substitute consisting of tricalcium phosphate composed of nanometer-sized pure &#946;-tricalcium phosphate (&#946;-TCP) particles and Type 1 bovine collagen (C) (&#946;-TCP/C foam) and 2) &#946;-TCP disks. Here, we illustrate biocompatibility testing of these biomaterials using primary osteoblast (OB) and osteoclast (OC) assays, an in vivo model of bone repair, an immunological assessment comprising in vitro T lymphocyte proliferation and cytokine production, and in vivo immunogenicity and allergenicity, as previously reported13.

<table><tbody><tr><td>&lt;strong&gt;Biomaterials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&amp;beta;-tricalcium phosphate disks (&amp;beta;-TCP, 14 mm)</td><td></td><td></td><td>Disks were sintered in a muffle furnace at a temperature of 1150 &amp;deg;C. A detailed description of the production can be found in Zimmer et al (doi: 10.1002/jbm.a.34639). Samples were UV-irradiated (15 min for each side) before using in cell cultures.</td></tr><tr><td>Orthovita Vitoss foam (&amp;beta;-TCP/C foam)</td><td>Orthovita</td><td>2102-1405</td><td></td></tr><tr><td>&lt;strong&gt;Mouse osteoblast culture&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Alizarin Red S</td><td>Sigma-Aldrich</td><td>A5533</td><td></td></tr><tr><td>ALP assay buffer (2x) pH 10.4</td><td></td><td></td><td>Dissolve 200 mM glycine, 2 mM magnesium chloride, 2 mM zinc chloride in ultrapure water and adjust to pH 10.</td></tr><tr><td>Bone growth medium (BGM)</td><td></td><td></td><td>&amp;alpha;-MEM + 10% heat-inactivated FBS + 1% penicillin/streptomycin</td></tr><tr><td>Cell lysis buffer: CyQuant cell lysis buffer 20x concentrate</td><td>Thermo Scientific</td><td>C7027</td><td>Dilute the concentrated cell lysis buffer stock 20-fold in ultrapure water.&amp;nbsp;</td></tr><tr><td>Cell viability reagent: Presto Blue cell viability reagent</td><td>Invitrogen, Thermo Fisher Scientific</td><td>A13261</td><td></td></tr><tr><td>Collagenase type IV from clostridium histolyticum</td><td>Sigma-Aldrich</td><td>C5138</td><td></td></tr><tr><td>DC protein assay kit II</td><td>Bio Rad</td><td>5000112</td><td>DC protein assay kit contains reagent A, reagent B, reagent S and BSA for the reference standard. For reagent A&#039;, add 20 &amp;micro;L reagent S to 1000 &amp;micro;L reagent A.&amp;nbsp;</td></tr><tr><td>Dimethylsulfoxide (DMSO)</td><td>Sigma-Aldrich</td><td>D8418</td><td>Steril-filter DMSO before usage.&amp;nbsp;</td></tr><tr><td>Dispase II (neutral protease, grade II)</td><td>Roche</td><td>4942078001</td><td></td></tr><tr><td>EnzCheck Phosphatase Assay Kit for alkaline phosphatase activity&amp;nbsp;</td><td>Invitrogen, Thermo Fisher Scientific</td><td>E12020</td><td>EnzCheck Phosphatase Assay Kit contains 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP), N,N-dimethylformamide and 6,8-difluoro-7-hydroxy-4-methylcoumarin (DiFMU).&amp;nbsp;</td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Sigma-Aldrich</td><td>F7524</td><td></td></tr><tr><td>Formalin solution neutral buffered 10%</td><td>Sigma-Aldrich</td><td>HT501128</td><td></td></tr><tr><td>Glycine</td><td>Sigma-Aldrich</td><td>G8898</td><td></td></tr><tr><td>Hexadecylpyrdinium (cetylpyridinium) chloride monohydrate</td><td>Sigma-Aldrich</td><td>C9002</td><td></td></tr><tr><td>L-Ascorbic acid 2-phosphate sesquimagnesium salt hydrate</td><td>Sigma-Aldrich</td><td>A8960</td><td></td></tr><tr><td>Magnesium chloride hexahydrate</td><td>Sigma-Aldrich</td><td>M2670</td><td></td></tr><tr><td>MEM alpha medium (&amp;alpha;-MEM)</td><td>Gibco Life Technologies&amp;nbsp;</td><td>11900-073</td><td></td></tr><tr><td>Osteogenic mineralization medium (MM)</td><td></td><td></td><td>&amp;alpha;-MEM + 10% heat-inactivated FBS + 1% penicillin-streptomycin + 50 &amp;micro;g/mL ascorbic acid + 5 mM &amp;beta;-glycerophosphate</td></tr><tr><td>Penicillin-streptomycin&amp;nbsp;&amp;nbsp;</td><td>Sigma-Aldrich</td><td>P4333</td><td></td></tr><tr><td>Phosphate-buffered saline 1x (PBS) pH 7.4</td><td>Gibco, Thermo Fisher Scientific&amp;nbsp;</td><td>10010023</td><td></td></tr><tr><td>Sigmafast BCIP/NBT</td><td>Sigma-Aldrich</td><td>B5655</td><td></td></tr><tr><td>Trypsin-EDTA (0.5%), no phenol red (10x)</td><td>Gibco, Thermo Fisher Scientific&amp;nbsp;</td><td>15400054</td><td>Dilute the concentrated stock solution 10-fold in 1x PBS.&amp;nbsp;</td></tr><tr><td>Tween20</td><td>Sigma-Aldrich</td><td>P1379</td><td></td></tr><tr><td>Ultrapure water: Cell culture water pyrogen free</td><td>VWR</td><td>L0970-500</td><td></td></tr><tr><td>Wash buffer</td><td></td><td></td><td>Add&amp;nbsp; 0.05% Tween20 to 1x PBS.</td></tr><tr><td>Zinc chloride</td><td>Sigma-Aldrich</td><td>1.08816</td><td></td></tr><tr><td>&amp;beta;-Glycerophosphate disodium salt hydrate</td><td>Sigma-Aldrich</td><td>G5422</td><td></td></tr><tr><td>24-well suspension culture plate</td><td>Greiner bio-one</td><td>662102</td><td></td></tr><tr><td>24-well tissue culture plate</td><td>Greiner bio-one</td><td>662160</td><td></td></tr><tr><td>50 mL conical tube</td><td>Greiner bio-one</td><td>227261</td><td></td></tr><tr><td>96-well black plate</td><td>Greiner bio-one</td><td>655076</td><td></td></tr><tr><td>96-well transparent plate</td><td>Greiner bio-one</td><td>655001</td><td></td></tr><tr><td>Centrifuge: VWR Mega Star 600R</td><td>VWR</td><td></td><td></td></tr><tr><td>CO2-Incubator: HeraCell 240</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>Cryovial 2 mL</td><td>Greiner bio-one</td><td>122263</td><td></td></tr><tr><td>Disposable filter unit FP30/0.2 CA-S</td><td>GE Healthcare Life Sciences Whatman</td><td>10462200</td><td>For sterile filtration of enzyme solution.</td></tr><tr><td>Flatbed scanner: Epson Perfection 1200 Photo</td><td>Epson</td><td></td><td></td></tr><tr><td>Infinite M200 Pro microplate reader</td><td>Tecan</td><td></td><td></td></tr><tr><td>Microcentrifuge tube: Eppendorf tube safe-lock 0.5 mL</td><td>VWR</td><td>20901-505</td><td></td></tr><tr><td>Microcentrifuge tube: Eppendorf tube safe-lock 1.5 mL</td><td>VWR</td><td>21008-959</td><td></td></tr><tr><td>Shaker Swip (orbital and horizontal)&amp;nbsp;</td><td>Edmund Buehler&amp;nbsp;</td><td></td><td></td></tr><tr><td>Shaking incubator GFL3032</td><td>GFL&amp;nbsp;</td><td>3032</td><td></td></tr><tr><td>Single-use pipet: serum pipet</td><td>Greiner bio-one</td><td>612301</td><td></td></tr><tr><td>Sterile instruments (scissors, tweezer, curved forceps)</td><td></td><td></td><td></td></tr><tr><td>Tissue culture plate (10 cm)</td><td>Greiner bio-one</td><td>664960</td><td></td></tr><tr><td>&lt;strong&gt;Mouse osteoblast-osteoclast co-culture&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>1&amp;alpha;,25-Dihydroxyvitamin D3</td><td>Sigma-Aldrich</td><td>17936</td><td>1000x concentrated stock (10 &amp;micro;M in ethanol 100%)</td></tr><tr><td>Acetone</td><td>VWR Chemicals</td><td>22065.327</td><td></td></tr><tr><td>Bone growth medium (BGM)</td><td></td><td></td><td>&amp;alpha;-MEM + 10% heat-inactivated FBS + 1% penicillin-streptomycin</td></tr><tr><td>Distilled water</td><td>Carl Roth</td><td>3478.4</td><td></td></tr><tr><td>Ethanol (100% vol/vol)</td><td>VWR Chemicals</td><td>20821.365</td><td></td></tr><tr><td>Ethanol (70% vol/vol)</td><td>VWR Chemicals</td><td>93003.1006</td><td></td></tr><tr><td>Fast red violet LB salt</td><td>Sigma-Aldrich</td><td>F3381</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Sigma-Aldrich</td><td>F7524</td><td></td></tr><tr><td>Formalin solution neutral buffered 10%</td><td>Sigma-Aldrich</td><td>HT501128</td><td></td></tr><tr><td>MEM alpha Medium (&amp;alpha;-MEM)</td><td>Gibco Life Technologies&amp;nbsp;</td><td>11900-073</td><td></td></tr><tr><td>N,N-Dimethylformamide</td><td>Sigma-Aldrich</td><td>D4551</td><td></td></tr><tr><td>Naphthol AS-MX phosphate</td><td>Sigma-Aldrich</td><td>N4875</td><td></td></tr><tr><td>Osteoclast differentiation medium (DM)</td><td></td><td></td><td>&amp;alpha;-MEM + 10% heat-inactivated FBS + 1% penicillin-streptomycin + 1 nM 1,25-(OH)2-vitamin D3 + 1 &amp;micro;M prostaglandin E2&amp;nbsp;</td></tr><tr><td>Penicillin-streptomycin&amp;nbsp;</td><td>Sigma-Aldrich</td><td>P4333</td><td></td></tr><tr><td>Phosphate-buffered saline 1x (PBS) pH 7.4</td><td>Gibco, Thermo Fisher Scientific&amp;nbsp;</td><td>10010023</td><td></td></tr><tr><td>Prostaglandin E2 (PGE2)</td><td>Cayman Chemical Company</td><td>9003016</td><td>1000x concentrated stock (1 mM in ethanol 100%)</td></tr><tr><td>Sodium acetate trihydrate</td><td>Sigma-Aldrich</td><td>S7670</td><td></td></tr><tr><td>Sodium tartrate dibasic dihydrate</td><td>Sigma-Aldrich</td><td>S8640</td><td></td></tr><tr><td>Sterile instruments (scissors, forceps, scalpel)</td><td></td><td></td><td></td></tr><tr><td>TRAP buffer pH 5.0</td><td></td><td></td><td>Dissolve 40 mM sodium acetate, 10 mM sodium tartrate in ultrapure water and adjust to pH 5.</td></tr><tr><td>Ultrapure water: Cell culture water pyrogen free</td><td>VWR</td><td>L0970-500</td><td></td></tr><tr><td>24-well suspension culture plate</td><td>Greiner bio-one</td><td>662102</td><td></td></tr><tr><td>24-well tissue culture plate</td><td>Greiner bio-one</td><td>662160</td><td></td></tr><tr><td>50 mL conical tube</td><td>Greiner bio-one</td><td>227261</td><td></td></tr><tr><td>Centrifuge: VWR Mega Star 600R</td><td>VWR</td><td></td><td></td></tr><tr><td>CO2-Incubator: HeraCell 240</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>Diamond wafering blade (10.2 cm x 0.3 cm)</td><td>Buehler</td><td></td><td></td></tr><tr><td>Isomet low-speed saw</td><td>Buehler</td><td></td><td></td></tr><tr><td>Petri dish (6 cm)</td><td>Greiner bio-one</td><td>628,161</td><td></td></tr><tr><td>Screw cap glass bottle 20 mL</td><td>Carl Roth</td><td>EXY4.1</td><td></td></tr><tr><td>Single-use sterile syringe 1 mL</td><td>Henry Schein Animal Health</td><td>9003016</td><td></td></tr><tr><td>Single-use pipet: serum pipet</td><td>Greiner bio-one</td><td>612301</td><td></td></tr><tr><td>Sterile needle: Hypodermic needle RW 27 G x 3/4&#039;&#039;</td><td>Henry Schein Animal Health</td><td>9003340</td><td></td></tr><tr><td>&lt;strong&gt;Mouse calvarial defect model&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Analgesia: Buprenorphine (Bupaq)</td><td>Richter Pharma AG</td><td></td><td></td></tr><tr><td>Anesthesia: Ketamine (Ketamidor), Xylazine (Rompun)</td><td>Richter Pharma AG/Bayer HealthCare</td><td></td><td></td></tr><tr><td>Cold sterilant: SafeSept Max</td><td>Henry Schein Animal Health</td><td>9882765</td><td></td></tr><tr><td>Ethanol (70% vol/vol)</td><td>VWR Chemicals</td><td>93003.1006</td><td></td></tr><tr><td>Eye ointment: VitA POS 5 g</td><td>Ursapharm</td><td></td><td></td></tr><tr><td>Normal saline solution (0.9% sodium chloride solution)</td><td>Fresenius Kabi</td><td></td><td></td></tr><tr><td>Phosphate-buffered saline 1x (PBS) pH 7.4</td><td>Gibco, Thermo Fisher Scientific&amp;nbsp;</td><td>10010023</td><td></td></tr><tr><td>Povidone iodine solution: Braunol 1000 mL</td><td>B. Braun</td><td>3864154</td><td></td></tr><tr><td>Roti-Histofix 4.5% buffered formalin</td><td>Carl Roth</td><td>2213.6</td><td></td></tr><tr><td>Tissue adhesive: Histoacryl 5 x 0.5 mL&amp;nbsp;</td><td>B. Braun Surgical</td><td>1050060</td><td></td></tr><tr><td>Personal protective equipment: surgical gloves, cap and mask, gown</td><td>Henry Schein Animal Health</td><td>1045073, 1026614, 9009062, 370406</td><td></td></tr><tr><td>Sterile instruments (scalpel, scissors, forceps, needle holder)</td><td></td><td></td><td></td></tr><tr><td>Sterile needles: Hypodermic needles RW 27 G x 3/4&#039;&#039; and 30 G x 1/2&#039;&#039;&amp;nbsp;</td><td>Henry Schein Animal Health</td><td>9003340, 9003630</td><td></td></tr><tr><td>Single-use sterile syringe 1 mL</td><td>Henry Schein Animal Health</td><td>9003016</td><td></td></tr><tr><td>Heating plate: Physitemp</td><td>Rothacher Medical</td><td>TCAT-2LV</td><td></td></tr><tr><td>Sterile gauze swabs</td><td>Henry Schein Animal Health</td><td>220192</td><td></td></tr><tr><td>Sterile disposable scalpel (Figur 20)</td><td>Henry Schein Animal Health</td><td>9008957</td><td></td></tr><tr><td>Surgical/dental drill: Implantmed SI-923</td><td>W&amp;amp;H Dentalwerk B&amp;uuml;rmoos GmbH</td><td>16929000</td><td></td></tr><tr><td>Handpiece type S-II</td><td>W&amp;amp;H Dentalwerk B&amp;uuml;rmoos GmbH</td><td>30056000</td><td></td></tr><tr><td>Trephine, diameter 4 mm</td><td>Hager&amp;amp;Meisinger GmbH</td><td>229040</td><td></td></tr><tr><td>Irrigation tubing set 2.2 m</td><td>W&amp;amp;H Dentalwerk B&amp;uuml;rmoos GmbH</td><td>4363600</td><td></td></tr><tr><td>Periosteal elevator 2 mm / 3 mm</td><td>Henry Schein Animal Health</td><td>472683</td><td></td></tr><tr><td>Non-resorbable suture: Polyester green, DS19, met. 1.5, USP 4/0 75 cm</td><td>Henry Schein Animal Health</td><td>300715</td><td></td></tr><tr><td>&lt;strong&gt;&lt;em&gt;In vitro&lt;/em&gt; immune responses&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Anesthesia for euthanasia: Ketamine (Ketamidor), Xylazine (Rompun)</td><td>Richter Pharma AG/Bayer HealthCare</td><td></td><td></td></tr><tr><td>Cell Proliferation ELISA, BrdU (colorimetric) Assay Kit</td><td>Sigma-Aldrich</td><td>11647229001</td><td>The kit contains BrdU labeling solution, fixation solution (FixDenat), Anti-BrdU antibody solution, washing buffer and substrate solution.&amp;nbsp;</td></tr><tr><td>Concanavalin A (ConA)</td><td>MP Biomedicals</td><td>150710</td><td></td></tr><tr><td>Culture medium splenocytes</td><td></td><td></td><td>RPMI medium + 10% heat-inactivated FBS + 1% penicillin-streptomycin solution + 0.1% gentamicin + 0.2% &amp;szlig;-mercaptoethanol + 1% non-essential amino acids</td></tr><tr><td>Ethanol (70% vol/vol)</td><td>VWR Chemicals</td><td>93003.1006</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Gibco, Thermo Fisher Scientific&amp;nbsp;</td><td>10500064</td><td></td></tr><tr><td>Gentamicin</td><td>Gibco, Thermo Fisher Scientific&amp;nbsp;</td><td>15750037</td><td></td></tr><tr><td>Mouse IL-1&amp;beta; ELISA Ready-SET-Go!</td><td>Invitrogen, Thermo Fisher Scientific</td><td>88-7013-88</td><td></td></tr><tr><td>Mouse IL-2 ELISA MAX Standard</td><td>BioLegend</td><td>431001</td><td></td></tr><tr><td>Mouse IL-4 ELISA MAX Standard</td><td>BioLegend</td><td>431101</td><td></td></tr><tr><td>Mouse INF-&amp;gamma; ELISA MAX Standard</td><td>BioLegend</td><td>430801</td><td></td></tr><tr><td>Non-essential amino acids solution</td><td>Gibco, Thermo Fisher Scientific&amp;nbsp;</td><td>11140050</td><td></td></tr><tr><td>Penicillin-streptomycin&amp;nbsp;</td><td>Gibco, Thermo Fisher Scientific&amp;nbsp;</td><td>15140122</td><td></td></tr><tr><td>Phosphate-buffered saline 1x (PBS) pH 7.4</td><td>Gibco, Thermo Fisher Scientific&amp;nbsp;</td><td>10010023</td><td></td></tr><tr><td>Red blood cell (RBC) lysis buffer: BD Pharm Lyse</td><td>BD Bioscience</td><td>555899</td><td></td></tr><tr><td>RPMI 1640 medium, HEPES</td><td>Gibco, Thermo Fisher Scientific&amp;nbsp;</td><td>52400025</td><td></td></tr><tr><td>&amp;beta;-Mercaptoethanol</td><td>Gibco, Thermo Fisher Scientific&amp;nbsp;</td><td>31350010</td><td></td></tr><tr><td>50 mL conical tube</td><td>Greiner bio-one</td><td>227261</td><td></td></tr><tr><td>96-well tissue culture plate</td><td>Greiner bio-one</td><td>655180</td><td></td></tr><tr><td>Centrifuge: VWR Mega Star 600R</td><td>VWR</td><td></td><td></td></tr><tr><td>Cell strainer 40 &amp;micro;m</td><td>BD Bioscience</td><td>352340</td><td></td></tr><tr><td>Infinite M200 Pro microplate reader</td><td>Tecan</td><td></td><td></td></tr><tr><td>Single-use sterile syringe 1 mL</td><td>Henry Schein Animal Health</td><td>9003016</td><td></td></tr><tr><td>Sterile surgical instruments (forceps, glass slides)</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;High throughput intraperitoneal model&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Anesthesia: Ketamine (Ketamidor), Xylazine (Rompun)</td><td>Richter Pharma AG/Bayer HealthCare</td><td></td><td></td></tr><tr><td>Cold sterilant: SafeSept Max</td><td>Henry Schein Animal Health</td><td>9882765</td><td></td></tr><tr><td>Ethanol (70% vol/vol)</td><td>VWR Chemicals</td><td>93003.1006</td><td></td></tr><tr><td>Eye ointment: VitA POS 5 g</td><td>Ursapharm</td><td></td><td></td></tr><tr><td>Mouse IL-1&amp;beta; ELISA Ready-SET-Go!</td><td>Invitrogen, Thermo Fisher Scientific</td><td>88-7013-88</td><td></td></tr><tr><td>Mouse IL-2 ELISA MAX Standard</td><td>BioLegend</td><td>431001</td><td></td></tr><tr><td>Mouse IL-4 ELISA MAX Standard</td><td>BioLegend</td><td>431101</td><td></td></tr><tr><td>Phosphate-buffered saline 1x (PBS) pH 7.4</td><td>Gibco, Thermo Fisher Scientific&amp;nbsp;</td><td>10010023</td><td></td></tr><tr><td>Povidone iodine solution: Braunol 1000 mL</td><td>B. Braun</td><td>3864154</td><td></td></tr><tr><td>Shandon Rapid-Chrome Kwik-Diff Staining Kit</td><td>Thermo Scientific</td><td>9990700</td><td>For differential cell count.</td></tr><tr><td>Heating plate: Physitemp</td><td>Rothacher Medical</td><td>TCAT-2LV</td><td></td></tr><tr><td>Hemocytometer: Neubauer chamber</td><td>Carl Roth</td><td>PC72.1</td><td></td></tr><tr><td>Non-resorbable suture: Ethibond Excel 4-0</td><td>Ethicon</td><td>6683H</td><td></td></tr><tr><td>Resorbable suture: Polysorb</td><td>Covidien</td><td>SL-5628</td><td></td></tr><tr><td>Shandon centrifuge for cytopsin</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>Single-use sterile syringe 1 mL</td><td>Henry Schein Animal Health</td><td>9003016</td><td></td></tr><tr><td>Sterile gauze swabs</td><td>Henry Schein Animal Health</td><td>220192</td><td></td></tr><tr><td>Sterile needles: Hypodermic needles RW 27 G x 3/4&#039;&#039; and 25 G</td><td>Henry Schein Animal Health</td><td>9003340, 420939</td><td></td></tr><tr><td>Sterile surgical instruments (scalpel, scissors, forceps, needle holder)</td><td></td><td></td><td></td></tr><tr><td>Trypan blue solution 0.4%</td><td>Gibco, Thermo Fisher Scientific&amp;nbsp;</td><td>15250061</td><td></td></tr><tr><td>&lt;strong&gt;Subchronic subcutaneous model&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Anesthesia: Ketamine (Ketamidor), Xylazine (Rompun)</td><td>Richter Pharma AG/Bayer HealthCare</td><td></td><td></td></tr><tr><td>Cold sterilant: SafeSept Max</td><td>Henry Schein Animal Health</td><td>9882765</td><td></td></tr><tr><td>Ethanol (70% vol/vol)</td><td>VWR Chemicals</td><td>93003.1006</td><td></td></tr><tr><td>Eye ointment: VitA POS 5 g</td><td>Ursapharm</td><td></td><td></td></tr><tr><td>Phosphate-buffered saline 1x (PBS) pH 7.4</td><td>Gibco, Thermo Fisher Scientific&amp;nbsp;</td><td>10010023</td><td></td></tr><tr><td>Povidone iodine solution: Braunol 1000 mL</td><td>B. Braun</td><td>3864154</td><td></td></tr><tr><td>Roti-Histofix 4.5% buffered formalin</td><td>Carl Roth</td><td>2213.6</td><td></td></tr><tr><td>Heating plate: Physitemp</td><td>Rothacher Medical</td><td>TCAT-2LV</td><td></td></tr><tr><td>Non-resorbable suture: Ethibond Excel 4-0</td><td>Ethicon</td><td>6683H</td><td></td></tr><tr><td>Personal protective equipment: surgical gloves, cap and mask, gown</td><td>Henry Schein Animal Health</td><td>1045073, 1026614, 9009062, 370406</td><td></td></tr><tr><td>Single-use sterile syringe 1 mL</td><td>Henry Schein Animal Health</td><td>9003016</td><td></td></tr><tr><td>Sterile gauze swabs</td><td>Henry Schein Animal Health</td><td>220192</td><td></td></tr><tr><td>Sterile instruments (scalpel, scissors, forceps, needle holder)</td><td></td><td></td><td></td></tr><tr><td>Sterile needle: Hypodermic needles RW 27 G x 3/4&#039;&#039;&amp;nbsp;</td><td>Henry Schein Animal Health</td><td>9003340, 420939</td><td></td></tr></tbody></table>

biological compatibility profile on biomaterials for bone regeneration

Large non-union bone fractures are a significant challenge in orthopedic surgery. Although auto- and allogeneic bone grafts are excellent for healing such lesions, there are potential complications with their use. Thus, material scientists are developing synthetic, biocompatible biomaterials to overcome these problems. In this study, we present a multidisciplinary platform for evaluating biomaterials for bone repair. We combined expertise from bone biology and immunology to develop a platform including in vitro osteoclast (OC) and osteoblast (OB) assays and in vivo mouse models of bone repair, immunogenicity, and allergenicity. We demonstrate how to perform the experiments, summarize the results, and report on biomaterial biocompatibility. In particular, we tested OB viability, differentiation, and mineralization and OC viability and differentiation in the context of &#946;-tricalcium phosphate (&#946;-TCP) disks. We also tested a &#946;-TCP/Collagen (&#946;-TCP/C) foam which is a commercially available material used clinically for bone repair in a critical-sized calvarial bone defect mouse model to determine the effects on the early phase of bone healing. In parallel experiments, we evaluated immune and allergic responses in mice. Our approach generates a biological compatibility profile of a bone biomaterial with a range of parameters necessary for predicting the biocompatibility of biomaterials used for bone healing and repair in patients.

To assess &#946;-TCP for its effectiveness as a biomaterial for bone repair, we used in vitro and in vivo screening methods. Firstly, we measured the OB responses to the &#946;-TCP disks compared with baseline medium alone controls. Figure 2 demonstrates the OB viability in response to &#946;-TCP disks at 7 and 14 days of culture. Cell viability measured from metabolically active cells in the culture wells was the same for OBs with medium in...

Watch this Scientific Journal Video about Biological Compatibility Profile on Biomaterials for Bone Regeneration at JoVE.com

Biological Compatibility Profile on Biomaterials for Bone Regeneration

The number of novel biomaterials engineered for repairing large bone lesions is continuously expanding with the aim to enhance bone healing and overcome the complications associated with bone transplantation. Here, we present a multidisciplinary strategy for pre-clinical biocompatibility testing of biomaterials for bone repair.

Large non-union bone fractures are a significant challenge in orthopedic surgery. Although auto- and allogeneic bone grafts are excellent for healing such ...

biological-compatibility-profile-on-biomaterials-for-bone-regeneration

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

12.0K Views.  University of Vienna. The number of novel biomaterials engineered for repairing large bone lesions is continuously expanding with the aim to enhance bone healing and overcome the complications associated with bone transplantation. Here, we present a multidisciplinary strategy for pre-clinical biocompatibility testing of biomaterials for bone repair.

Video: Biological Compatibility Profile on Biomaterials for Bone Regeneration

An Improved Mechanical Testing Method to Assess Bone-implant Anchorage

Recent advances in material science have led to a substantial increase in the topographical complexity of implant surfaces, both on a micro- and a nano-scale. As such, traditional methods of describing implant surfaces -&#160;namely numerical determinants of surface roughness -&#160;are inadequate for predicting in vivo performance. Biomechanical testing provides an accurate and comparative platform to analyze the performance of biomaterial surfaces. An improved mechanical testing method to test the anchorage of bone to candidate implant surfaces is presented. The method is applicable to both early and later stages of healing and can be employed for any range of chemically or mechanically modified surfaces -&#160;but not smooth surfaces. Custom rectangular implants are placed bilaterally in the distal femora of male Wistar rats and collected with the surrounding bone. Test specimens are prepared and potted using a novel breakaway mold and the disruption test is conducted using a mechanical testing machine. This method allows for alignment of the disruption force exactly perpendicular, or parallel, to the plane of the implant surface, and provides an accurate and reproducible means for isolating an exact peri-implant region for testing.

An improved method to mechanically test bone anchorage to candidate implant surfaces is presented. This method allows for alignment of the disruption force exactly perpendicular, or parallel, to the plane of the implant surface, and provides an accurate means to direct the disruption forces to an exact peri-implant region.

Recent advances in material science have led to a substantial increase in the topographical complexity of implant surfaces, both on a micro- and a ...

Distinctive Capillary Action by Micro-channels in Bone-like Templates can Enhance Recruitment of Cells for Restoration of Large Bony Defect

Without an active, thriving cell population that is well-distributed and stably anchored to the inserted template, exceptional bone regeneration does not occur. With conventional templates, the absence of internal micro-channels results in the lack of cell infiltration, distribution, and inhabitance deep inside the templates. Hence, a highly porous and uniformly interconnected trabecular-bone-like template with micro-channels (biogenic microenvironment template; BMT) has been developed to address these obstacles. The novel BMT was created by innovative concepts (capillary action) and fabricated with a sponge-template coating technique. The BMT consists of several structural components: inter-connected primary-pores (300-400 &#181;m) that mimic pores in trabecular bone, micro-channels (25-70 &#181;m) within each trabecula, and nanopores (100-400 nm) on the surface to allow cells to anchor. Moreover, the BMT has been documented by mechanical test study to have similar mechanical strength properties to those of human trabecular bone (~3.8 MPa)12.
The BMT exhibited high absorption, retention, and habitation of cells throughout the bridge-shaped (&#928;) templates (3 cm height and 4 cm length). The cells that were initially seeded into one end of the templates immediately mobilized to the other end (10 cm distance) by capillary action of the BMT on the cell media. After 4 hr, the cells homogenously occupied the entire BMT and exhibited normal cellular behavior. The capillary action accounted for the infiltration of the cells suspended in the media and the distribution (active migration) throughout the BMT. Having observed these capabilities of the BMT, we project that BMTs will absorb bone marrow cells, growth factors, and nutrients from the periphery under physiological conditions.
The BMT may resolve current limitations via rapid infiltration, homogenous distribution and inhabitance of cells in large, volumetric templates to repair massive skeletal defects.

A step-by-step generic process to create a bone-like template with engineered micro-channels is presented. High absorption and retention capabilities of the template are demonstrated by capillary action via micro-channels.

Without an active, thriving cell population that is well-distributed and stably anchored to the inserted template, exceptional bone regeneration does not ...

Site-Directed Immobilization of Bone Morphogenetic Protein 2 to Solid Surfaces by Click Chemistry

Different therapeutic strategies for the treatment of non-healing long bone defects have been intensively investigated. Currently used treatments present several limitations that have led to the use of biomaterials in combination with osteogenic growth factors, such as bone morphogenetic proteins (BMPs). Commonly used absorption or encapsulation methods require supra-physiological amounts of BMP2, typically resulting in a so-called initial burst release effect that provokes several severe adverse side effects. A possible strategy to overcome these problems would be to covalently couple the protein to the scaffold. Moreover, coupling should be performed in a site-specific manner in order to guarantee a reproducible product outcome. Therefore, we created a BMP2 variant, in which an artificial amino acid (propargyl-L-lysine) was introduced into the mature part of the BMP2 protein by codon usage expansion (BMP2-K3Plk). BMP2-K3Plk was coupled to functionalized beads through copper catalyzed azide-alkyne cycloaddition (CuAAC). The biological activity of the coupled BMP2-K3Plk was proven in vitro and the osteogenic activity of the BMP2-K3Plk-functionalized beads was proven in cell based assays. The functionalized beads in contact with C2C12 cells were able to induce alkaline phosphatase (ALP) expression in locally restricted proximity of the bead. Thus, by this technique, functionalized scaffolds can be produced that can trigger cell differentiation towards an osteogenic lineage. Additionally, lower BMP2 doses are sufficient due to the controlled orientation of site-directed coupled BMP2. With this method, BMPs are always exposed to their receptors on the cell surface in the appropriate orientation, which is not the case if the factors are coupled via non-site-directed coupling techniques. The product outcome is highly controllable and, thus, results in materials with homogeneous properties, improving their applicability for the repair of critical size bone defects.

Biomaterials doped with Bone Morphogenetic Protein 2 (BMP2) have been used as a new therapeutic strategy to heal non-union bone fractures. To overcome side effects resulting from an uncontrollable release of the factor, we propose a new strategy to site-directly immobilize the factor, thus creating materials with improved osteogenic capabilities.

Different therapeutic strategies for the treatment of non-healing long bone defects have been intensively investigated. Currently used treatments present ...

Ceramic Omnidirectional Bioprinting in Cell-Laden Suspensions for the Generation of Bone Analogs

Structurally, bone tissue is an inorganic-organic composite containing metabolically active cells embedded within a hierarchical, highly mineralized matrix. This organization is challenging to replicate due to the heterogeneous environment of bone. Ceramic omnidirectional bioprinting in cell-suspensions (COBICS) is a microgel-based bioprinting technique that uniquely replicates the mineral and cellular structure of bone. COBICS prints complex, biologically relevant constructs without the need for sacrificial support materials or harsh postprocessing steps (e.g., radiation and high-temperature sintering), which are two of the biggest challenges in the additive manufacturing of bone mimetic constructs. This technique is enabled via the freeform extrusion of a novel calcium phosphate-based ink within a gelatin-based microgel suspension. The yield-stress properties of the suspension allow deposition and support the printed bone structure. UV crosslinking and nanoprecipitation then "lock" it in place. The ability to print nanostructured bone-mimetic ceramics within cell-laden biomaterials provides spatiotemporal control over macro- and micro-architecture and facilitates the real-time fabrication of complex bone constructs in clinical settings.

This protocol describes a 3D printing technique to fabricate bone-like structures by depositing a calcium phosphate ink in a gelatin-based granular support. Printed bone analogs are deposited in freeform, with flexibility for direct harvesting of the print or crosslinking within a living cell matrix for multiphasic constructs.

Structurally, bone tissue is an inorganic-organic composite containing metabolically active cells embedded within a hierarchical, highly mineralized ...

Oral Biofilm Formation on Different Materials for Dental Implants

Dental implants and their prosthetic components are prone to bacterial colonization and biofilm formation. The use of materials that provides low microbial adhesion may reduce the prevalence and progression of peri-implant diseases. In view of the oral environment complexity and oral biofilm heterogeneity, microscopy techniques are needed that can enable a biofilm analysis of the surfaces of teeth and dental materials. This article describes a series of protocols implemented for comparing oral biofilm formation on titanium and ceramic materials for prosthetic abutments, as well as the methods involved in oral biofilms analyses at the morphological and cellular levels. The in situ model to evaluate oral biofilm formation on titanium and zirconia materials for dental prosthesis abutments as described in this study provides a satisfactory preservation of the 48 h biofilm, thereby demonstrating methodological adequacy. Multiphoton microscopy allows the analysis of an area representative of the biofilm formed on the test materials. In addition, the use of fluorophores and the processing of the images using multiphoton microscopy allows the analysis of the bacterial viability in a very heterogeneous population of microorganisms. The preparation of biological specimens for electron microscopy promotes the structural preservation of biofilm, images with good resolution, and no artifacts.

Here, we present a protocol to evaluate oral biofilm formation on titanium and zirconia materials for dental prosthesis abutments, including the analysis of bacterial cells viability and morphological characteristics. An in situ model associated with powerful microscopy techniques is used for the oral biofilm analysis.

Dental implants and their prosthetic components are prone to bacterial colonization and biofilm formation. The use of materials that provides low ...

Medicine

Analyzing Cell-Seeded Decellularized Apple-Derived Scaffolds for Bone Tissue Engineering

Decellularized Apple-Derived Scaffolds for Bone Tissue Engineering In Vitro and In Vivo

Plant-derived&#160;cellulose biomaterials have been employed in various tissue engineering applications. In vivo studies have shown the remarkable biocompatibility of scaffolds made of cellulose derived from natural sources. Additionally, these scaffolds possess structural characteristics that are relevant for multiple tissues, and they promote the invasion and proliferation of mammalian cells. Recent research using decellularized apple hypanthium tissue has demonstrated the similarity of its pore size to that of trabecular bone as well as&#160;its ability to effectively support osteogenic differentiation. The present study further&#160;examined the potential of apple-derived cellulose scaffolds for bone tissue engineering (BTE) applications and evaluated their in vitro and in vivo mechanical properties. MC3T3-E1 preosteoblasts were seeded in apple-derived cellulose scaffolds that were then&#160;assessed for their osteogenic potential and mechanical properties. Alkaline phosphatase and alizarin red S staining confirmed osteogenic differentiation in scaffolds cultured in differentiation medium. Histological examination demonstrated widespread cell invasion and mineralization across the scaffolds. Scanning electron microscopy (SEM) revealed mineral aggregates on the surface of the scaffolds, and&#160;energy-dispersive spectroscopy (EDS)&#160;confirmed the presence of phosphate and calcium elements. However, despite a significant increase in the Young&#39;s modulus following cell differentiation, it remained lower than that of healthy bone tissue. In vivo studies showed cell infiltration and deposition of extracellular matrix within the decellularized apple-derived&#160;scaffolds after 8 weeks of implantation in rat calvaria. In addition, the force required to remove the scaffolds from the bone defect was similar to the previously reported fracture load of native calvarial bone. Overall, this study confirms that apple-derived cellulose is a promising candidate for BTE applications. However, the dissimilarity between its mechanical properties and those of healthy bone tissue may restrict its application to low load-bearing scenarios. Additional structural re-engineering and optimization may be necessary to enhance the mechanical properties of apple-derived cellulose scaffolds for load-bearing applications.

Author Spotlight: Insights into the Use of Apple-Derived Cellulose Scaffolds for Bone Tissue Engineering 

In this study, we detail methods of decellularization, physical characterization, imaging, and in vivo implantation of plant-based biomaterials, as well as methods for cell seeding and differentiation in the scaffolds. The described methods allow the evaluation of plant-based biomaterials for bone tissue engineering applications.

Plant-derived&#160;cellulose biomaterials have been employed in various tissue engineering applications. In vivo studies have shown the remarkable ...

Multimodal Analysis of Bone Formation Using Explant Models

Comprehensive Characterization of Tissue Mineralization in an Ex Vivo Model

The extensive characterization of tissue mineralization in the context of bone regeneration represents a significant challenge, given the numerous modalities that are currently available for analysis. Here, we propose a workflow for a comprehensive evaluation of new bone formation using a relevant large animal osseous ex vivo explant. A bone defect (diameter = 3.75 mm; depth = 5.0 mm) is created in an explanted sheep femoral head and injected with a macroporous bone substitute loaded with a pro-osteogenic growth factor (bone morphogenetic protein 2 - BMP2). Subsequently, the explant is maintained in culture for a 28-day period, allowing cellular colonization and subsequent bone formation. To evaluate the quality and structure of newly mineralized tissue, the following successive methods are set up: (i) Characterization and high-resolution 3D images of the entire explant using micro-CT, followed by deep learning image analyses to enhance the discrimination of mineralized tissues; (ii) Nano-indentation to determine the mechanical properties of the newly formed tissue; (iii) Histological examinations, such as Hematoxylin/Eosin/Saffron (HES), Goldner's trichrome, and Movat's pentachrome to provide a qualitative assessment of mineralized tissue, particularly with regard to the visualization of the osteoid barrier and the presence of bone cells; (iv) Back-scattering scanning electron microscopy (SEM) mapping with internal reference to quantify the degree of mineralization and provide detailed insights into surface morphology, mineral composition, and bone-biomaterial interface; (v) Raman spectroscopy to characterize the molecular composition of the mineralized matrix and to provide insights into the persistence of BMP2 within the cement through the detection of peptide bonds. This multimodal analysis will provide an effective assessment of newly formed bone and comprehensive qualitative and quantitative insights into mineralized tissues. Through the standardization of these protocols, we aim to facilitate interstudy comparisons and improve the validity and reliability of research findings.

Author Spotlight: Advanced Techniques for Characterizing Tissue Mineralization in Bone Regeneration Research

The proposed protocol entails a global approach to assess bone formation in the context of bone regeneration using multimodal analyses. It aims to provide qualitative and quantitative information on new bone formation, enhancing the rigor and validity of basic and pre-clinical investigations.

The extensive characterization of tissue mineralization in the context of bone regeneration represents a significant challenge, given the numerous ...

Accessing the Cytotoxicity and Cell Response to Biomaterials

Biomaterials contact directly or indirectly with the human tissues, making it important to evaluate its cytotoxicity. This evaluation can be performed by several methods, but a high discrepancy exists between the approaches used, compromising the reproducibility and the comparison among the obtained results. In this paper, we propose a protocol to evaluate biomaterials cytotoxicity using soluble extracts, which we use for dental biomaterials. The extracts preparation is detailed, from pellets production to its extraction in a culture medium. The biomaterials cytotoxicity evaluation is based on metabolic activity using the MTT assay, cell viability using the Sulphorhodamine B (SBR) assay, cell death profile by flow cytometry, and cell morphology using May-Gr&#252;nwald Giemsa. Additional to cytotoxicity evaluation, a protocol to evaluate cell function is described based on the expression of specific markers assessed by immunocytochemistry and PCR. This protocol provides a comprehensive guide for biomaterials cytotoxicity and cellular effects evaluation, using the extracts methodology, in a reproducible and robust manner.

This methodology aims to evaluate biomaterial cytotoxicity through the preparation of soluble extracts, using viability assays and phenotypic analysis, including flow cytometry, RT-PCR, immunocytochemistry, and other cellular and molecular biology techniques.

Biomaterials contact directly or indirectly with the human tissues, making it important to evaluate its cytotoxicity. This evaluation can be performed by ...

Bone Conditioned Medium: Preparation and Bioassay

Autologous bone grafts are widely used in oral and maxillofacial surgery, orthopedics, and traumatology. Autologous bone grafts not only replace missing bone, they also support the complex process of bone regeneration. This favorable behavior of autografts is attributed to the three characteristics: osteoconductivity, osteogenicity, and osteoinductivity. However, there is another aspect: Bone grafts release a myriad of molecules, including growth factors, which can target mesenchymal cells involved in bone regeneration. The paracrine properties of bone grafts can be studied in vitro by the use of bone-conditioned medium (BCM). Here we present a protocol on how to prepare bone-conditioned medium from native pig cortical bone, and bone that underwent thermal processing or demineralization. Cells can be directly exposed to BCM or seeded onto biomaterials, such as collagen membranes, previously soaked with BCM. We give examples for in vitro bioassays with mesenchymal cells on the expression of TGF-&#946; regulated genes. The presented protocols should encourage to further reveal the paracrine effects of bone grafts during bone regeneration and open a path for translational research in the broad field of reconstructive surgery.

We describe here how to prepare bone-conditioned medium (BCM) and test its activity in vitro.

Autologous bone grafts are widely used in oral and maxillofacial surgery, orthopedics, and traumatology. Autologous bone grafts not only replace missing ...

Biology

Overview of Biomaterials

Biomaterials are materials engineered to interact favorably with biological organisms or molecules. These materials can be derived from or produced by an organism, or can even be a synthesized polymer. Engineers use these novel materials in a wide range of applications, such as tissue engineering, biosensing and drug delivery.
This video introduces common biologically derived materials, and provides examples of common techniques used to process them. Key challenges in the field are discussed, along with several applications of these methods.

Biomaterials: Chitosan, Silk; Biocompatibility and Biodegradation

Biomaterials are materials engineered to interact favorably with biological organisms or molecules. These materials can be derived from or produced by an ...