Antallet af roman biomaterialer manipuleret til at reparere store knogle læsioner løbende udvide med formålet at forbedre knogleheling og overvinde komplikationer forbundet med knogle transplantation. Vi præsenterer her, en tværfaglig strategi for prækliniske biokompatibilitet testning af biomaterialer for knogle reparation.
Store ikke-union knoglebrud er en betydelig udfordring i ortopædisk kirurgi. Selv om auto- og allogene bone podninger er fremragende for at helbrede sådan læsioner, er der potentielle komplikationer med deres anvendelse. Således, materiale forskere udvikler syntetisk, biokompatible biomaterialer for at overvinde disse problemer. I denne undersøgelse præsenterer vi en tværfaglig platform for evaluering af biomaterialer for knogle reparation. Vi kombinerede ekspertise fra knogle biologi og Immunologi at udvikle en platform, herunder in vitro- osteoklast (OC) og osteoblastdannelse (OB) assays og i vivo musemodeller af knogle reparation, immunogenicitet og allergenicitet. Vi demonstrere, hvordan at udføre eksperimenter, opsummere resultaterne og rapportere om biomateriale biokompatibilitet. Navnlig, testede vi OB levedygtighed, differentiering, og mineralisering og OC levedygtighed og differentiering i forbindelse med β-tricalciumphosphat fosfat (β-TCP) diske. Vi har også testet en β-TCP/kollagen (β-TCP/C) skum, som er et kommercielt tilgængelige materiale bruges klinisk til knogle reparation i en kritisk størrelse calvarial knogle defekt musemodel for at fastslå virkningerne på den tidlige fase af knogleheling. I parallel eksperimenter evalueret vi immun og allergiske reaktioner hos mus. Vores tilgang skaber en biologisk forenelighed profil af en knogle biomateriale med en række parametre, der er nødvendige for at forudsige biokompatibilitet af biomaterialer bruges til knogleheling og reparation hos patienter.
Knogle reparation er en kompleks proces, der begynder med hæmatom dannelse, betændelse, callus dannelse og derefter remodeling1,2. Knogle regenerering potentielle er dog begrænset til størrelsen af knogle fraktur1,3. For eksempel, store knoglebrud forårsaget af traumer, kræft eller osteoporose kan ikke helbrede og kaldes ikke-union knoglebrud. Disse knogler læsioner kræver ofte behandling for at fremme sunde fysiologiske knogle reparation og regeneration. I øjeblikket, autograft og allograft knogle transplantation er tilgangen i valg4 med 2,2 millioner knogle udskiftning procedurer årligt5. Selvom disse procedurer har en høj succesrate, kan der være komplikationer, for eksempel, begrænset tilgængelighed af knoglen, infektion, donor site sygelighed og afvisning4. Nye alternativer til knogle vævsmanipulering er søges til at løse disse udfordringer.
Design af biomaterialer baseret på naturlige eller syntetiske polymerer, bioceramics eller metaller i kombination med celler og bioaktive molekyler er på rise6. Vores nuværende forståelse af fysiologiske knogle heling og helbredelse i forbindelse med biomaterialer afhænger af flere faktorer som mekaniske egenskaber og flere lokale og systemiske faktorer herunder celler fra omsætning og fraktur site7 ,8,9. Biomaterialer for knogle regenerering har til formål at fremme osteogenicity og osseointegration10 og er ideelt biokompatible, biologisk nedbrydeligt og porøse (fremme celle migration, ilt og næringsstoffer). De skal også være tilstrækkeligt stærke til at støtte fraktur websted for at lindre smerter. Derudover er provokerende faktorer forpligtet til at indlede helingsprocessen. Men hvis en biomateriale inducerer overdreven betændelse og allergiske reaktioner, kan det begrænse eller hæmme knogle heling11,12. Tværfaglighed er således nødvendigt at vurdere biomaterialer udviklet til knogle reparation.
I denne undersøgelse præsenterer vi en præ-klinisk evaluering af repræsentative materialer, 1) Orthovita Vitoss skum, som er et kommercielt tilgængelige spongiosa graft erstatning bestående af tricalciumphosphat sammensat af nanometer-størrelse ren β-tricalciumphosphat fosfat (β-TCP) partikler og Type 1 bovine kollagen (C) (β-TCP/C skum) og 2) β-TCP diske. Her, vi illustrere biokompatibilitet testning af disse biomaterialer ved hjælp af primær osteoblastdannelse (OB) og osteoklast (OC) undersøgelser, en i vivo model af knogle reparation, immunologiske vurdering bestående af in vitro- T-lymfocytter spredning og cytokin produktion, og i vivo immunogenicitet og allergenicitet, som tidligere rapporteret13.
Her viser vi en tværfaglig tilgang til prækliniske vurdering af biokompatibilitet for repræsentative biomaterialer udviklet til knogle regenerering og reparation. Vi testede svarene fra OBs, OCs, og in vivo helbredende reaktion i en kritisk knogle defekt model i mus samt in vitro og i vivo immunrespons. Vi havde til formål at demonstrere hvordan assays arbejde og opsummere data og konklusioner stammer fra en undersøgelse af biomaterialerne. Vi viser, at vores strategi genererer et værdifu…
The authors have nothing to disclose.
Dette forskningsprojekt har modtaget støtte fra EUs syvende rammeprogram (FP7/2007-2013) under grant aftale nr. 263363.
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 |