Perfil de compatibilidad biológica de Biomateriales para regeneración ósea
Summary
El número de nuevos biomateriales ingeniería para reparar las lesiones del hueso grande está en continua expansión con el objetivo de acelerar la cicatrización ósea y superar las complicaciones asociadas con el trasplante de hueso. Aquí, presentamos una estrategia multidisciplinaria para las pruebas de biocompatibilidad preclínico de Biomateriales para la reparación de hueso.
Abstract
Fracturas del hueso grande de la Unión no son un reto importante en cirugía ortopédica. Aunque auto e injertos óseos Alogénicos son excelentes para la curación de estas lesiones, existen posibles complicaciones con su uso. Así, materiales científicos están desarrollando biomateriales sintéticos, biocompatibles para superar estos problemas. En este estudio, presentamos una plataforma multidisciplinar para la evaluación de Biomateriales para la reparación de hueso. Combinamos conocimientos de Biología ósea y de la inmunología para desarrollar una plataforma como en vitro de los osteoclastos (OC) y osteoblastos (OB) ensayos en vivo modelos de ratón de reparación ósea, inmunogenicidad y alergenicidad. Nos muestran cómo realizar los experimentos, resumir los resultados e informar sobre biocompatibilidad de los biomateriales. En particular, hemos probado OB viabilidad, diferenciación y mineralización y viabilidad OC y diferenciación en el contexto de los discos de β-tricálcico fosfato (β-TCP). También probamos una espuma de β-TCP/colágeno (β-TCP/C) que es un material disponible comercialmente utilizado clínicamente para la reparación ósea en un modelo de ratón de defecto hueso calvarial tamaño crítico para determinar los efectos en la fase temprana de la curación del hueso. En experimentos paralelos, se evaluaron las respuestas inmunes y alérgicas en ratones. Nuestro enfoque genera un perfil de compatibilidad biológica de un biomaterial de hueso con una serie de parámetros necesarios para la predicción de la biocompatibilidad de los biomateriales utilizados para la curación del hueso y la reparación en pacientes.
Introduction
Reparación ósea es un proceso complejo que comienza con la formación de hematoma, inflamación, formación y remodelación luego1,2de callos. Sin embargo, potencial de la regeneración ósea se limita al tamaño de la fractura ósea1,3. Por ejemplo, las fracturas del hueso grande causadas por trauma, cáncer u osteoporosis no pueden curar y se denominan fracturas sin unión. Estas lesiones del hueso a menudo requieren tratamiento para promover la regeneración y reparación ósea fisiológica sana. Actualmente, el trasplante de autoinjerto y aloinjerto óseo es el enfoque de opción4 con procedimientos de reemplazo de hueso 2,2 millones anualmente5. Aunque estos procedimientos tienen una alta tasa de éxito, puede haber complicaciones, por ejemplo, la disponibilidad limitada de hueso, la infección, la morbosidad del sitio donante y la rechazo4. Se buscan nuevas alternativas para la ingeniería de tejido óseo para enfrentar estos desafíos.
El diseño de biomateriales basados en polímeros naturales o sintéticos, biocerámica o metales en combinación con las células y moléculas bioactivas está en el lugar6. Nuestra comprensión actual del hueso fisiológico sanación y sanación en el contexto de biomateriales depende de múltiples factores tales como propiedades mecánicas y de múltiples factores locales y sistémicos, incluyendo las células de la circulación y el sitio de la fractura7 ,8,9. Biomateriales para regeneración ósea intentan promover osteogenicity y osteointegración10 y son idealmente biocompatible, biodegradable y poroso (promueve la migración celular, nutrientes y oxígeno). También necesitan ser lo suficientemente fuerte para soportar el sitio de la fractura para aliviar el dolor. Además, factores inflamatorios están obligados a iniciar el proceso de curación. Sin embargo, si el biomaterial induce inflamación excesiva y las respuestas alérgicas, esto podría limitar o inhibir el hueso curativo11,12. Por lo tanto, un enfoque interdisciplinario es necesario evaluar biomateriales desarrollados para la reparación de hueso.
En este estudio, presentamos una evaluación preclínica de los materiales representativos, 1) Orthovita Vitoss espuma que es un sustituto de injerto de hueso esponjoso disponible en el mercado compuesto por fosfato tricálcico se compone de β-tricálcico puro tamaño de nanómetros partículas de fosfato (β-TCP) y colágeno bovino tipo 1 (C) (espuma de β-TCP/C) y 2) discos de β-TCP. Aquí ilustramos pruebas de biocompatibilidad de los biomateriales con osteoblastos primarios (OB) y ensayos de los osteoclastos (OC), un modelo en vivo de la reparación de hueso, una evaluación inmunológica que en vitro proliferación de linfocitos T y producción de citoquinas y en vivo inmunogenicidad y alergenicidad, como previamente divulgados13.
Protocol
Representative Results
Discussion
A continuación, os mostramos un enfoque multidisciplinario para la evaluación preclínica de la biocompatibilidad de biomateriales representativos desarrollado para la reparación y regeneración ósea. Probamos las respuestas de OBs, OCs, y la respuesta curativa en vivo en un defecto óseo importante modelo en ratones, así como in vitro e in vivo las respuestas inmunitarias. El objetivo fue demostrar cómo los análisis del trabajan y resumen los datos y conclusiones derivadas de la examina…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Este proyecto de investigación ha recibido financiación del séptimo programa de marco de la Unión Europea (FP7/2007-2013) con beca Convenio núm. 263363.
Materials
| Biomaterials | |||
| β-tricalcium phosphate disks (β-TCP, 14 mm) | Disks were sintered in a muffle furnace at a temperature of 1150 °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. | ||
| Orthovita Vitoss foam (β-TCP/C foam) | Orthovita | 2102-1405 | |
| Mouse osteoblast culture | |||
| Alizarin Red S | Sigma-Aldrich | A5533 | |
| ALP assay buffer (2x) pH 10.4 | Dissolve 200 mM glycine, 2 mM magnesium chloride, 2 mM zinc chloride in ultrapure water and adjust to pH 10. | ||
| Bone growth medium (BGM) | α-MEM + 10% heat-inactivated FBS + 1% penicillin/streptomycin | ||
| Cell lysis buffer: CyQuant cell lysis buffer 20x concentrate | Thermo Scientific | C7027 | Dilute the concentrated cell lysis buffer stock 20-fold in ultrapure water. |
| Cell viability reagent: Presto Blue cell viability reagent | Invitrogen, Thermo Fisher Scientific | A13261 | |
| Collagenase type IV from clostridium histolyticum | Sigma-Aldrich | C5138 | |
| DC protein assay kit II | Bio Rad | 5000112 | DC protein assay kit contains reagent A, reagent B, reagent S and BSA for the reference standard. For reagent A', add 20 µL reagent S to 1000 µL reagent A. |
| Dimethylsulfoxide (DMSO) | Sigma-Aldrich | D8418 | Steril-filter DMSO before usage. |
| Dispase II (neutral protease, grade II) | Roche | 4942078001 | |
| EnzCheck Phosphatase Assay Kit for alkaline phosphatase activity | Invitrogen, Thermo Fisher Scientific | E12020 | EnzCheck Phosphatase Assay Kit contains 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP), N,N-dimethylformamide and 6,8-difluoro-7-hydroxy-4-methylcoumarin (DiFMU). |
| Fetal bovine serum (FBS) | Sigma-Aldrich | F7524 | |
| Formalin solution neutral buffered 10% | Sigma-Aldrich | HT501128 | |
| Glycine | Sigma-Aldrich | G8898 | |
| Hexadecylpyrdinium (cetylpyridinium) chloride monohydrate | Sigma-Aldrich | C9002 | |
| L-Ascorbic acid 2-phosphate sesquimagnesium salt hydrate | Sigma-Aldrich | A8960 | |
| Magnesium chloride hexahydrate | Sigma-Aldrich | M2670 | |
| MEM alpha medium (α-MEM) | Gibco Life Technologies | 11900-073 | |
| Osteogenic mineralization medium (MM) | α-MEM + 10% heat-inactivated FBS + 1% penicillin-streptomycin + 50 µg/mL ascorbic acid + 5 mM β-glycerophosphate | ||
| Penicillin-streptomycin | Sigma-Aldrich | P4333 | |
| Phosphate-buffered saline 1x (PBS) pH 7.4 | Gibco, Thermo Fisher Scientific | 10010023 | |
| Sigmafast BCIP/NBT | Sigma-Aldrich | B5655 | |
| Trypsin-EDTA (0.5%), no phenol red (10x) | Gibco, Thermo Fisher Scientific | 15400054 | Dilute the concentrated stock solution 10-fold in 1x PBS. |
| Tween20 | Sigma-Aldrich | P1379 | |
| Ultrapure water: Cell culture water pyrogen free | VWR | L0970-500 | |
| Wash buffer | Add 0.05% Tween20 to 1x PBS. | ||
| Zinc chloride | Sigma-Aldrich | 1.08816 | |
| β-Glycerophosphate disodium salt hydrate | Sigma-Aldrich | G5422 | |
| 24-well suspension culture plate | Greiner bio-one | 662102 | |
| 24-well tissue culture plate | Greiner bio-one | 662160 | |
| 50 mL conical tube | Greiner bio-one | 227261 | |
| 96-well black plate | Greiner bio-one | 655076 | |
| 96-well transparent plate | Greiner bio-one | 655001 | |
| Centrifuge: VWR Mega Star 600R | VWR | ||
| CO2-Incubator: HeraCell 240 | Thermo Scientific | ||
| Cryovial 2 mL | Greiner bio-one | 122263 | |
| Disposable filter unit FP30/0.2 CA-S | GE Healthcare Life Sciences Whatman | 10462200 | For sterile filtration of enzyme solution. |
| Flatbed scanner: Epson Perfection 1200 Photo | Epson | ||
| Infinite M200 Pro microplate reader | Tecan | ||
| Microcentrifuge tube: Eppendorf tube safe-lock 0.5 mL | VWR | 20901-505 | |
| Microcentrifuge tube: Eppendorf tube safe-lock 1.5 mL | VWR | 21008-959 | |
| Shaker Swip (orbital and horizontal) | Edmund Buehler | ||
| Shaking incubator GFL3032 | GFL | 3032 | |
| Single-use pipet: serum pipet | Greiner bio-one | 612301 | |
| Sterile instruments (scissors, tweezer, curved forceps) | |||
| Tissue culture plate (10 cm) | Greiner bio-one | 664960 | |
| Mouse osteoblast-osteoclast co-culture | |||
| 1α,25-Dihydroxyvitamin D3 | Sigma-Aldrich | 17936 | 1000x concentrated stock (10 µM in ethanol 100%) |
| Acetone | VWR Chemicals | 22065.327 | |
| Bone growth medium (BGM) | α-MEM + 10% heat-inactivated FBS + 1% penicillin-streptomycin | ||
| Distilled water | Carl Roth | 3478.4 | |
| Ethanol (100% vol/vol) | VWR Chemicals | 20821.365 | |
| Ethanol (70% vol/vol) | VWR Chemicals | 93003.1006 | |
| Fast red violet LB salt | Sigma-Aldrich | F3381 | |
| Fetal bovine serum (FBS) | Sigma-Aldrich | F7524 | |
| Formalin solution neutral buffered 10% | Sigma-Aldrich | HT501128 | |
| MEM alpha Medium (α-MEM) | Gibco Life Technologies | 11900-073 | |
| N,N-Dimethylformamide | Sigma-Aldrich | D4551 | |
| Naphthol AS-MX phosphate | Sigma-Aldrich | N4875 | |
| Osteoclast differentiation medium (DM) | α-MEM + 10% heat-inactivated FBS + 1% penicillin-streptomycin + 1 nM 1,25-(OH)2-vitamin D3 + 1 µM prostaglandin E2 | ||
| Penicillin-streptomycin | Sigma-Aldrich | P4333 | |
| Phosphate-buffered saline 1x (PBS) pH 7.4 | Gibco, Thermo Fisher Scientific | 10010023 | |
| Prostaglandin E2 (PGE2) | Cayman Chemical Company | 9003016 | 1000x concentrated stock (1 mM in ethanol 100%) |
| Sodium acetate trihydrate | Sigma-Aldrich | S7670 | |
| Sodium tartrate dibasic dihydrate | Sigma-Aldrich | S8640 | |
| Sterile instruments (scissors, forceps, scalpel) | |||
| TRAP buffer pH 5.0 | Dissolve 40 mM sodium acetate, 10 mM sodium tartrate in ultrapure water and adjust to pH 5. | ||
| Ultrapure water: Cell culture water pyrogen free | VWR | L0970-500 | |
| 24-well suspension culture plate | Greiner bio-one | 662102 | |
| 24-well tissue culture plate | Greiner bio-one | 662160 | |
| 50 mL conical tube | Greiner bio-one | 227261 | |
| Centrifuge: VWR Mega Star 600R | VWR | ||
| CO2-Incubator: HeraCell 240 | Thermo Scientific | ||
| Diamond wafering blade (10.2 cm x 0.3 cm) | Buehler | ||
| Isomet low-speed saw | Buehler | ||
| Petri dish (6 cm) | Greiner bio-one | 628,161 | |
| Screw cap glass bottle 20 mL | Carl Roth | EXY4.1 | |
| Single-use sterile syringe 1 mL | Henry Schein Animal Health | 9003016 | |
| Single-use pipet: serum pipet | Greiner bio-one | 612301 | |
| Sterile needle: Hypodermic needle RW 27 G x 3/4'' | Henry Schein Animal Health | 9003340 | |
| Mouse calvarial defect model | |||
| Analgesia: Buprenorphine (Bupaq) | Richter Pharma AG | ||
| Anesthesia: Ketamine (Ketamidor), Xylazine (Rompun) | Richter Pharma AG/Bayer HealthCare | ||
| Cold sterilant: SafeSept Max | Henry Schein Animal Health | 9882765 | |
| Ethanol (70% vol/vol) | VWR Chemicals | 93003.1006 | |
| Eye ointment: VitA POS 5 g | Ursapharm | ||
| Normal saline solution (0.9% sodium chloride solution) | Fresenius Kabi | ||
| Phosphate-buffered saline 1x (PBS) pH 7.4 | Gibco, Thermo Fisher Scientific | 10010023 | |
| Povidone iodine solution: Braunol 1000 mL | B. Braun | 3864154 | |
| Roti-Histofix 4.5% buffered formalin | Carl Roth | 2213.6 | |
| Tissue adhesive: Histoacryl 5 x 0.5 mL | B. Braun Surgical | 1050060 | |
| Personal protective equipment: surgical gloves, cap and mask, gown | Henry Schein Animal Health | 1045073, 1026614, 9009062, 370406 | |
| Sterile instruments (scalpel, scissors, forceps, needle holder) | |||
| Sterile needles: Hypodermic needles RW 27 G x 3/4'' and 30 G x 1/2'' | Henry Schein Animal Health | 9003340, 9003630 | |
| Single-use sterile syringe 1 mL | Henry Schein Animal Health | 9003016 | |
| Heating plate: Physitemp | Rothacher Medical | TCAT-2LV | |
| Sterile gauze swabs | Henry Schein Animal Health | 220192 | |
| Sterile disposable scalpel (Figur 20) | Henry Schein Animal Health | 9008957 | |
| Surgical/dental drill: Implantmed SI-923 | W&H Dentalwerk Bürmoos GmbH | 16929000 | |
| Handpiece type S-II | W&H Dentalwerk Bürmoos GmbH | 30056000 | |
| Trephine, diameter 4 mm | Hager&Meisinger GmbH | 229040 | |
| Irrigation tubing set 2.2 m | W&H Dentalwerk Bürmoos GmbH | 4363600 | |
| Periosteal elevator 2 mm / 3 mm | Henry Schein Animal Health | 472683 | |
| Non-resorbable suture: Polyester green, DS19, met. 1.5, USP 4/0 75 cm | Henry Schein Animal Health | 300715 | |
| In vitro immune responses | |||
| Anesthesia for euthanasia: Ketamine (Ketamidor), Xylazine (Rompun) | Richter Pharma AG/Bayer HealthCare | ||
| Cell Proliferation ELISA, BrdU (colorimetric) Assay Kit | Sigma-Aldrich | 11647229001 | The kit contains BrdU labeling solution, fixation solution (FixDenat), Anti-BrdU antibody solution, washing buffer and substrate solution. |
| Concanavalin A (ConA) | MP Biomedicals | 150710 | |
| Culture medium splenocytes | RPMI medium + 10% heat-inactivated FBS + 1% penicillin-streptomycin solution + 0.1% gentamicin + 0.2% ß-mercaptoethanol + 1% non-essential amino acids | ||
| Ethanol (70% vol/vol) | VWR Chemicals | 93003.1006 | |
| Fetal bovine serum (FBS) | Gibco, Thermo Fisher Scientific | 10500064 | |
| Gentamicin | Gibco, Thermo Fisher Scientific | 15750037 | |
| Mouse IL-1β ELISA Ready-SET-Go! | Invitrogen, Thermo Fisher Scientific | 88-7013-88 | |
| Mouse IL-2 ELISA MAX Standard | BioLegend | 431001 | |
| Mouse IL-4 ELISA MAX Standard | BioLegend | 431101 | |
| Mouse INF-γ ELISA MAX Standard | BioLegend | 430801 | |
| Non-essential amino acids solution | Gibco, Thermo Fisher Scientific | 11140050 | |
| Penicillin-streptomycin | Gibco, Thermo Fisher Scientific | 15140122 | |
| Phosphate-buffered saline 1x (PBS) pH 7.4 | Gibco, Thermo Fisher Scientific | 10010023 | |
| Red blood cell (RBC) lysis buffer: BD Pharm Lyse | BD Bioscience | 555899 | |
| RPMI 1640 medium, HEPES | Gibco, Thermo Fisher Scientific | 52400025 | |
| β-Mercaptoethanol | Gibco, Thermo Fisher Scientific | 31350010 | |
| 50 mL conical tube | Greiner bio-one | 227261 | |
| 96-well tissue culture plate | Greiner bio-one | 655180 | |
| Centrifuge: VWR Mega Star 600R | VWR | ||
| Cell strainer 40 µm | BD Bioscience | 352340 | |
| Infinite M200 Pro microplate reader | Tecan | ||
| Single-use sterile syringe 1 mL | Henry Schein Animal Health | 9003016 | |
| Sterile surgical instruments (forceps, glass slides) | |||
| High throughput intraperitoneal model | |||
| Anesthesia: Ketamine (Ketamidor), Xylazine (Rompun) | Richter Pharma AG/Bayer HealthCare | ||
| Cold sterilant: SafeSept Max | Henry Schein Animal Health | 9882765 | |
| Ethanol (70% vol/vol) | VWR Chemicals | 93003.1006 | |
| Eye ointment: VitA POS 5 g | Ursapharm | ||
| Mouse IL-1β ELISA Ready-SET-Go! | Invitrogen, Thermo Fisher Scientific | 88-7013-88 | |
| Mouse IL-2 ELISA MAX Standard | BioLegend | 431001 | |
| Mouse IL-4 ELISA MAX Standard | BioLegend | 431101 | |
| Phosphate-buffered saline 1x (PBS) pH 7.4 | Gibco, Thermo Fisher Scientific | 10010023 | |
| Povidone iodine solution: Braunol 1000 mL | B. Braun | 3864154 | |
| Shandon Rapid-Chrome Kwik-Diff Staining Kit | Thermo Scientific | 9990700 | For differential cell count. |
| Heating plate: Physitemp | Rothacher Medical | TCAT-2LV | |
| Hemocytometer: Neubauer chamber | Carl Roth | PC72.1 | |
| Non-resorbable suture: Ethibond Excel 4-0 | Ethicon | 6683H | |
| Resorbable suture: Polysorb | Covidien | SL-5628 | |
| Shandon centrifuge for cytopsin | Thermo Scientific | ||
| Single-use sterile syringe 1 mL | Henry Schein Animal Health | 9003016 | |
| Sterile gauze swabs | Henry Schein Animal Health | 220192 | |
| Sterile needles: Hypodermic needles RW 27 G x 3/4'' and 25 G | Henry Schein Animal Health | 9003340, 420939 | |
| Sterile surgical instruments (scalpel, scissors, forceps, needle holder) | |||
| Trypan blue solution 0.4% | Gibco, Thermo Fisher Scientific | 15250061 | |
| Subchronic subcutaneous model | |||
| Anesthesia: Ketamine (Ketamidor), Xylazine (Rompun) | Richter Pharma AG/Bayer HealthCare | ||
| Cold sterilant: SafeSept Max | Henry Schein Animal Health | 9882765 | |
| Ethanol (70% vol/vol) | VWR Chemicals | 93003.1006 | |
| Eye ointment: VitA POS 5 g | Ursapharm | ||
| Phosphate-buffered saline 1x (PBS) pH 7.4 | Gibco, Thermo Fisher Scientific | 10010023 | |
| Povidone iodine solution: Braunol 1000 mL | B. Braun | 3864154 | |
| Roti-Histofix 4.5% buffered formalin | Carl Roth | 2213.6 | |
| Heating plate: Physitemp | Rothacher Medical | TCAT-2LV | |
| Non-resorbable suture: Ethibond Excel 4-0 | Ethicon | 6683H | |
| Personal protective equipment: surgical gloves, cap and mask, gown | Henry Schein Animal Health | 1045073, 1026614, 9009062, 370406 | |
| Single-use sterile syringe 1 mL | Henry Schein Animal Health | 9003016 | |
| Sterile gauze swabs | Henry Schein Animal Health | 220192 | |
| Sterile instruments (scalpel, scissors, forceps, needle holder) | |||
| Sterile needle: Hypodermic needles RW 27 G x 3/4'' | Henry Schein Animal Health | 9003340, 420939 | |
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