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

The procedures were done with BALB/c mice following all guidelines for the Care and Use of Laboratory Animals of the Austrian Ministry of Education, Science and Research and were approved by the Committee on the Ethics of the Austrian Ministry of Education, Science and Research.
1. Primary Mouse OB Culture
<ol>
	<li>OB isolation from neonatal mouse calvaria using enzymatic digestion
	<ol>
		<li>Euthanize 1&#8211;2 day old neonatal pups (60 in total) by decapitation and place...

<ol>
	<li>Einhorn, T. A., Gerstenfeld, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fracture+healing:+mechanisms+and+interventions.">Fracture healing: mechanisms and interventions.</a> Nature Reviews Rheumatology. 11 (1), 45-54 (2015).</li><li>Marsell, R., Einhorn, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biology+of+fracture+healing.">The biology of fracture healing.</a> Injury. 42 (6), 551-555 (2011).</li><li>Cooper, G. M., 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=Testing+the+critical+size+in+calvarial+bone+defects:+revisiting+the+concept+of+a+critical-size+defect.">Testing the critical size in calvarial bone defects: revisiting the concept of a critical-size defect.</a> Plastic and Reconstructive Surgery. 125 (6), 1685-1692 (2010).</li><li>O'Brien, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomaterials+&amp;+scaffolds+for+tissue+engineering.">Biomaterials &amp; scaffolds for tissue engineering.</a> Materials Today. 14 (3), 88-95 (2011).</li><li>Stanovici, J., 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=Bone+regeneration+strategies+with+bone+marrow+stromal+cells+in+orthopaedic+surgery.">Bone regeneration strategies with bone marrow stromal cells in orthopaedic surgery.</a> Current Research in Translational Medicine. 64 (2), 83-90 (2016).</li><li>Anderson, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Future+challenges+in+the+in+vitro+and+in+vivo+evaluation+of+biomaterial+biocompatibility.">Future challenges in the in vitro and in vivo evaluation of biomaterial biocompatibility.</a> Regenerative Biomaterials. 3 (2), 73-77 (2016).</li><li>Chen, Z., 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=Osteoimmunomodulation+for+the+development+of+advanced+bone+biomaterials.">Osteoimmunomodulation for the development of advanced bone biomaterials.</a> Materials Today. 19 (6), 304-321 (2016).</li><li>Ono, T., Takayanagi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteoimmunology+in+bone+fracture+healing.">Osteoimmunology in bone fracture healing.</a> Current Osteoporosis Reports. 15 (4), 367-375 (2017).</li><li>Tsiridis, E., Upadhyay, N., Giannoudis, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+aspects+of+fracture+healing:+Which+are+the+important+molecules?.">Molecular aspects of fracture healing: Which are the important molecules?.</a> Injury. 38, S11-S25 (2007).</li><li>Velasco, M. A., Narvaez-Tovar, C. A., Garzon-Alvarado, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design,+materials,+and+mechanobiology+of+biodegradable+scaffolds+for+bone+tissue+engineering.">Design, materials, and mechanobiology of biodegradable scaffolds for bone tissue engineering.</a> BioMed Research International. 2015, 729076 (2015).</li><li>Bastian, O., 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=Systemic+inflammation+and+fracture+healing.">Systemic inflammation and fracture healing.</a> Journal of Leukocyte Biology. 89 (5), 669-673 (2011).</li><li>El-Jawhari, J. J., Jones, E., Giannoudis, P. 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. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beta+tricalcium+phosphate:+observation+of+use+in+100+posterolateral+lumbar+instrumented+fusions.">Beta tricalcium phosphate: observation of use in 100 posterolateral lumbar instrumented fusions.</a> Spine Journal. 9 (8), 630-638 (2009).</li><li>Kallai, I., 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=Microcomputed+tomography-based+structural+analysis+of+various+bone+tissue+regeneration+models.">Microcomputed tomography-based structural analysis of various bone tissue regeneration models.</a> Nature Protocols. 6 (1), 105-110 (2011).</li><li>Lo, D. 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. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+matrix-induced+osteogenesis+in+rat+calvarial+bone+defects:+II.+Origins+of+bone-forming+cells.">Characterization of matrix-induced osteogenesis in rat calvarial bone defects: II. Origins of bone-forming cells.</a> Calcified Tissue International. 65 (6), 486-493 (1999).</li></ol>

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.

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 ...

These methods can help answer the key questions in the field of biomaterials. Particularly for bone regeneration. They're used to evaluate the effectiveness of biomaterials and potential immune responses.The main advantage of this multidisciplinary platform is that it provides a range of parameters for predicting biocompatibility of biomaterials used for bone repair. The implications of these in vitro and in vivo bone techniques extend towards other bone disorders. Due to the need for preclinical screening of novel orthopedic biomaterials.The dermatological methodology can provide insights into biomaterials for bone healing, it can also be applied to biomaterial assessments for any other clinical indication. Aspirate culture medium from primary osteoblasts 14 days after the addition of osteogenic mineralization medium. Rinse twice with 0.5 milliliters of 1X PBS.Then fix the cells by adding 0.5 milliliters of 10%buffered formalin solution and incubating at room temperature for 10 minutes. After 10 minutes, aspirate the 10%buffered formalin with a single-use pipet and wash twice with 0.5 milliliters of ultrapure water. Then add 250 microliters of 40 millimolar Alizarin Red S staining solution to the fixed osteoblasts.And incubate the plate at room temperature for 10 minutes on a shaker at 100 shakes per minute. Following the incubation with staining solution, aspirate the staining solution with a single-use pipet and rinse with one milliliter of ultrapure water. Repeat this step five to 10 times until the rinsing solution comes off clear to remove nonspecific staining.After aspirating the final wash, add one milliliter of cold PBS. And incubate the plate at room temperature for 10 minutes on a shaker as before. Next, transfer the stained beta tricalcium phosphate discs to a new well and scan the plates with a flatbed scanner to record mineralization.After image capture, add 250 microliters of 10%cetylpyridinium chloride solution and shake for 15 minutes to extract the Alizarin Red S dye. Next, transfer the solution from the wells to individual 1.5 milliliters tubes and centrifuge at 17, 000 x g for five minutes at room temperature. Dilute the extract with 10%cetylpyridinium chloride solution at a dilution ration of 1:10 to 1:20 in a 1.5 milliliter tube.Then transfer 300 microliters of each diluted sample into the wells of the 96-well plate. Include two blank wells containing only 10%cetylpyridinium chloride. Next, prepare seven Alizarin Red S reference standards ranging from 4 400 micromolar by diluting 40 milliomolar Alizarin Red S staining solution with 10%cetylpyridinium chloride solution to generate a standard curve.Load 300 microliters of each standard into the plate. Lastly, read the absorbents of the samples, blanks, and reference standards at 520 nanometers. Isolate bone marrow osteoplast precursors from the femurs and tibiae of a euthanized mouse by using sterile scissors to remove the legs from the body at the hip joint.Cut the limbs at the knee and ankle joints and remove the soft tissue with sterile scalpel and forceps. Cut off the epiphyses. Insert a 27 gauge needle attached to a syringe filled with one milliliter of bone growth medium into the lumen and flush out the marrow into a six centimeter sterile Petri dish.After repeating this for all femurs and tibiae, transfer the cell suspension to a 50 milliliter conical tube. Wash the six centimeter Petri dish with five milliliters of bone growth medium and transfer it to the same 50 milliliter conical tube. Centrifuge at 350 x g at four degrees Celsius for five minutes.Following centrifugation re-suspend the cells in one milliliter per well of osteoclast differentiation medium. One BALB/c mouse provides sufficient numbers of osteoclast precursors for one 24-well culture plate. Add primary osteoblasts suspended in bone growth medium onto the biomaterial and the controls at 8.8*10^4 cells per square centimeter.Beta tricalcium phosphate discs, controlled bovine bone, and tissue culture plastic are used here. Incubate at 37 degrees celsius in five percent carbon dioxide for 24 hours. After 24 hours, add freshly isolated bone marrow osteoclast precursors and osteoclast differentiation medium to the cultured primary osteoblasts and return to the incubator for five days of culture at 37 degrees celsius at five percent carbon dioxide.Replace the medium with freshly-prepared osteoclast differentiation medium every other day. Then stain osteoclasts for tartrate resistant acid phosphatase to evaluate differentiation. Shave the scalp of an anesthetized eight-week-old BALB/c mouse and clean the surface with 7.5%povidone iodine solution and 70%ethanol.Cover the eyes with ophthalmic ointment to prevent drying during the procedure. Ensure an appropriate level of anesthesia by lack of a reaction to a toe and tail pinch. Then after making a midline sagittal incision remove the paracranium connective tissues above the right parietal bone by scraping it with a scalpel.Then use a sterile dental trephine with a diameter of four millimeters at 2000 rotations per minute to create a critical-sized defect in the right parietal bone. Constantly irrigate the region with sterile saline solution while notching the right parietal bone and cutting through the ectocortext and some endocortex. To prevent damage to the Dera Mater, use a small periosteal elevator to break through the remaining endocortex.Then use forceps to carefully lift the calvarial bone and remove it. The resulting defect should be circular and approximately 3.5 millimeters in diameter. Next, fill the calvarial defect with beta tricalcium phosphate collagen foam, presoaked in sterile PBS.To keep the biomaterial in place, apply 0.5 microliters of tissue adhesive at the end of the defect at two opposite points. Then close the skin with a nonabsorbable suture. Place an anesthetized mouse on its back, shave the abdominal fur and clean the shaved surface with 7.5%povidone iodine solution and 70%ethanol.After making an eight millimeter-long midline incision through the skin along the linea alba of the abdomen, implant PBS soaked biomaterial under the skin. Then suture the skin with a nonabsorbable suture. Eight weeks post-implantation, excise the implantation site with surrounding tissue from the euthanized mouse.This image demonstrates that growth on beta tricalcium phosphate discs shown in open bars does not affect osteoblast viability compared to growth on tissue culture plastic at seven and 14 days of culture. Cultured osteoblasts were stained with Alizarin Red S after 14 days. Mineralization was higher for mineralization medium controls compared to osteoblasts cultured in medium alone.And on beta tricalcium phosphate discs in suspension culture wells. When a surgically-induced critical-sized calvarial defect was left empty, a thin layer covering the entire defect was observed, but no significant bone formation was present at 12 weeks post-operation. In contrast, when the defect contained beta tricalcium phosphate collagen foam, foam remnants surrounded by dense fibrous tissue including some blood vessels and inflammatory cells bridged the defect area without evidence of bone formation.Following subcutaneous beta tricalcium phosphate collagen foam implantation, an inflammatory response with foreign body giant cells was observed on hematoxylin and Eosin-stained sections an evidence of fibrosis on Masson's trichrome stained sections at eight weeks. In contrast, the implantation site of the sham controls had minimal inflammation and no fibrosis. While attempting this procedure it's important to do precise and reproducible notching of the calvarium without hurting the animal.Following this procedure's other methods like osteoclast differentiations assays and high throughput into peritoneal immune models can be preformed in order to answer additional questions like osteoclast's response to biomaterials and type of immune response.

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11.8K ビュー.  University of Vienna. これらの方法は、生体材料の分野における重要な質問に答えるのに役立ちます。特に骨再生のため。彼らは、生体材料の有効性と潜在的な免疫応答を評価するために使用されます。この学際的なプラットホームの主な利点は骨の修理のために使用される生物材料の生物適合性を予測するための変数の範囲を提供する。これらのインビトロおよびインビボ骨技術の意?...