Abstract
Objective
To study the in vivo osteoinductive potential, bone-bonding ability (bioactivity) and bone biomineralization of current hydraulic calcium silicate cements used as graft materials and placed in contact with medullary bone.
Methods
ProRoot MTA, MTA Plus and Biodentine were used to fill surgical bone defects (2-mm diameter through the entire cortical thickness to reach the medullary bone) in the tibia of mature male rabbits. Tibiae were retrieved after 30 days and submitted to histological analysis and microchemical characterization using Optical Microscopy (OM) and Environmental Scanning Electron Microscopy with Energy Dispersive X-ray analysis (ESEM-EDX). Bone neoformation and histomorphometric evaluations, degree of mineralization (by Ca/P, Ca/N and P/N ratios) and the diffusion of material elements were studied.
Results
Bone neoformation was observed in response to all materials. No sign of necrosis were found on the walls of the pre-existing cortical bone. No osteoclasts and no formation of fibrous tissue were evident. Sign of angiogenesis were present.
EDX (element content, line profile and element mapping) showed the increase in Ca and P and decrease in C, S and N from the mature bone towards the mineralizing interface.
Ca/P, Ca/N and P/N ratios showed differences in the degree of mineralization/maturation stage of bone.
MTA Plus and ProRoot MTA exhibited close contact with the pre-existing bone and good bone-bonding with neoformed bone juxtaposed on the medullary side of the materials without interposed connective tissue or resorption lacunae or gaps. The materials showed a dense appearance with 100% of residual materials and no colonization by fluids and cells. No migration of Bi or Al material elements to the newly formed bone was found.
Biodentine showed newly formed trabecular bone with marrow spaces and sparse traces of residual material (≈9%).
Significance
The in vivo osteoinductive properties with dynamic biomineralization processes around these calcium silicate materials extruded in medullary bone in appropriate animal model have been demonstrated by ESEM-EDX in association with OM. Good biocompatibility was evident as only slight inflammatory infiltrate and no sign of necrosis at the interface with the pre-existing bone were found.
MTA Plus and ProRoot MTA exhibited bioactive potential as they can bond to bone directly without interposed connective tissue. Biodentine was replaced by newly formed bone.
Clinical significance
The results of the study demonstrate the capacity of calcium silicate cements to allow osteoid matrix deposition by activated osteoblasts and favour its biomineralization, and to achieve a direct bond between the (bioactive) materials surface and the mineralized bone matrix.
1
Introduction
Root-end filling materials are placed in contact with the medullary alveolar bone when used to seal resected root apices, to plug root perforations and to fill wide open apices. Wide open apices in nonvital immature teeth and in over-instrumented root canals with a resected apex need the orthograde placement of appropriate sealing materials as apical barrier/plug . A critical point is the biological response of the periapical medullary bone, as large amounts of material can be extruded into the periapical tissue when used to plug wide apices and root perforations, producing toxic effects, (additional) tissue inflammation and foreign body reactions. Therefore, the use of biocompatible osteoconductive (preferably osteoinductive) materials is advocated to avoid extrusion-related complications or the impairment of the bone healing process.
Currently, hydraulic calcium silicate cements represent the golden standard among the materials for root-end filling to seal/close the root in root apex resection, root perforation repair and apexification in relationship to their specific and suitable chemical–physical (they are non resorbable materials setting in fluid/blood contaminated environments having slight degradability and low solubility ) and biointeractive properties correlatable with their positive biological effect . Their ability to release biologically relevant ions and the property to nucleate calcium phosphates/apatite suggest their pivotal role in mineral tissue regeneration by activating the osteogenic potential and promoting the differentiation of mineralizing-cells as human bone marrow stromal cells, human orofacial bone marrow mesenchymal stem cells and osteoblasts .
Previous histological studies on the intraosseous placement of hydraulic calcium silicate cements in different animal models showed contradictory results.
Bone healing and minimal inflammatory response adjacent to the implants were observed as response to freshly mixed ProRoot MTA or Portland cement inserted (inside cylindrical Teflon applicators, 2 mm diameter and 2 mm length) into the bone cavities in guinea pigs mandible .
A toxicity level diminishing with elapsing time and excellent biological qualities with bone growth in close contact with the material and no interposing connective tissue has been reported for ProRoot MTA implanted in the lower jaw symphysis of guinea-pigs .
Differently, a mild-to-moderate chronic inflammatory cell infiltration consisting of lymphocytes, macrophages, fibroblasts, and some giant cells was present in a thin fibrous capsule generated at 30 days as response to MTA Angelus inside polyethylene tubes in rat alveolar sockets . The fibrous capsule was present near the tube was thin, and the bone tissue with dystrophic calcification was close to the material . The intensity of the inflammation reduced with time and was absent at 90 days.
Similarly, bone tissue reaction to ProRoot MTA filling implantation cavities studied in a rat femur model showed a decrease of the number of inflammatory cells together with the increase of the new bone formation with the implantation time .
Otherwise, both ProRoot MTA and an experimental dicalcium silicate implanted in distal mesial rabbit femur incorporated well with the surrounding tissue and exhibited no inflammatory response, rejection or necrosis in the adjacent host tissue .
Bone healing and minimal inflammatory response adjacent to MTAs implanted in proximal rabbit femur were observed at 3–12 weeks .
MTAs and Portland cement implanted in rabbit mandible showed bone healing and regeneration .
Experimental calcium silicates cements implanted in bone defects in rabbit tibiae showed bone repairing capacity and osteoconductive potential .
In the present study current hydraulic calcium silicate cements (Biodentine, MTA Plus and ProRoot MTA) have been used as graft materials in bone defects and intentionally placed in contact with the medullary cavity through the entire cortical thickness of rabbit tibia.
The histological analysis associated with the microchemical characterization by ESEM-EDX have been performed to study their osteoinductive potential and bone-bonding ability (bioactivity) and the morphostructural features of the healing bone tissue at the interface with cancellous/medullary bone.
2
Materials and methods
2.1
Surgical procedure
Skeletally mature (9 month old, 3.5 kg weigh) pathogen free (SPF) and virus antibody free (VAF) male New Zealand white rabbits (Crl:KBL(NZW)) obtained from Charles River Laboratories (Lieu-dit Oncins, France) were used. The protocol conformed to the guiding principles of ISO 10993-2 (Animal welfare requirements) and ISO 10993-1 (Part 6: tests for local effects after implantation) and was approved by the Ethical Committee of University of Chieti-Pescara, Italy.
The rabbits were anesthetized with intramuscular injections of fluanizone (0.7 mg/kg body weight) and diazepam (1.5 mg/kg body weight), and local anaesthesia was given using 1 mL of 2% lidocain/adrenalin solution. The medial surface of the tibiae was exposed via a skin excision with a periosteal flap . Care was taken to split the muscular layer by blunt dissection and to keep the periosteum intact apart from the longitudinal excision. Cylindrical noncritical-sized bone defects (2 mm in diameter) were performed in the medial diaphyseal face of rabbit tibia using a 2 mm diameter drill working at 300 rpm under constant copious saline irrigation. The bone excision was performed through the entire cortical thickness in order to reach the medullary space. Defects were filled with Biodentine (Septodont, Saint-Maur-des-Fossés, France; batch number B01767), MTA Plus with gel (Prevest Detpro Limited, Jammu, India; lot n. 41001) or ProRoot MTA (Dentsply Tulsa, Johnson City, TN, USA; batch number 09003850) as graft materials (n = 6 for each material in 6 different animals, following ISO 10993-1) . All cements were prepared following the manufacturer directions and inserted manually into the bone defects using a stainless steel spatula, and extruded into the medullary area. In the control group the noncritical-sized defects were left empty and were allowed to heal spontaneously.
The overlying soft tissues, periostium and fascia, were sutured with catgut and the skin with silk suture. Eight bone defects were created in each animal, 4 in the right tibia, and 4 in the left tibia ( Fig. 1 ).
Oxytetracycline dihydrate (Terramicina long Acting by Pfizer Italia srl) 100 mg/kg single dose and analgesics with tramadol hydrochloride (Altadol Abiogen Pharma S.p.A Italia) were given for 1 week. Sutures were removed 2 weeks after surgery. Postsurgical visits were scheduled daily to check the course of healing. No complications or deaths occurred in the postoperative period.
The animals were pharmacologically euthanized 30 days after surgery (following ISO 10993-1 for prolonged-permanent long-term contact of implant devices for bone tissue) with an overdose of intravenous Tanax (Intervet Italia srl, Peschiera Borromeo, Mi, Italy) under general anaesthesia with intramuscular injections of fluanizone (0.7 mg/kg body weight) and diazepam (1.5 mg/kg body weight). Then, the bone samples were retrieved and processed for histological analysis.
2.2
Histological specimen processing
The retrieved specimens were immediately stored in 10% buffered formalin and processed to obtain thin ground sections with the Precise 1 Automated System (Assing, Rome, Italy) . They were dehydrated in an ascending series of alcohol rinses and embedded in a glycolmethacrylate resin (Technovit 7200, VLC, Kulzer, Wehrheim, Germany). After polymerization, the specimens were sectioned longitudinally along the major axis of the rabbit tibiae with a high-precision diamond disc at about 150 μm and ground down to about 30 μm. Then, the slides were stained with acid fuchsin and toluidine blue.
2.3
Histological analysis
2.3.1
EDX microchemical analysis and ESEM examination
The histological sections were examined using an environmental scanning electron microscope (ESEM, Zeiss EVO 50; Carl Zeiss, Oberkochen, Germany) connected to a secondary electron detector for energy dispersive X-ray analysis (EDX; Oxford INCA 350 EDS, Abingdon, UK) using computer-controlled software (Inca Energy Version 18). The sections were examined uncoated at low vacuum (100 Pascal), 20 kV accelerating voltage, 8.5 mm working distance, 0.5 wt% detection level, 133 eV resolution, 100 microseconds amplification time, measuring time: 600 s for element mapping and 60 s for spectra. The resulting electron beam penetration inside was approx. 2 μm.
ESEM histological observations were performed at 100×, 1500× and 3000× magnification.
EDX microchemical analysis (elemental X-ray microanalysis) was carried out at random in areas of approx. 50 × 50 μm to evaluate the relative element content. Microanalysis (weight % and atomic %) with ZAF correction method was performed in full frame and spot mode to analyze entire areas or specific points respectively. The Ca/P, Ca/N and P/N ratios were calculated from the data to evaluate the degree of mineralization of the newly formed bone.
EDX element mapping was performed at the bone-material interface to detect the element distribution within the mature and neoformed bone and the presence in the surrounding tissues of elements from the implanted material.
Element mapping was performed using 512 × 384 pixel matrix, 30–40 frames, 100 μs dwelling time, 600–700 total reading time.
Line profile (line scans) through the bone-interface-material were performed to detect the variation content of Ca, P, S, C and N from the mature pre-existing bone towards the implanted material.
Scan line was carried out using 300 s reading time.
2.3.2
OM examination and histomorphometry
Histological analysis and histomorphometry of the percentage of newly formed bone, marrow spaces and residual biomaterial was carried out using a light microscope (Leitz Laborlux, Wetzlar, Germany) connected to a high resolution video camera (3CCD, JVC KY-F55B, JVC, Yokohama, Japan) and interfaced to a monitor and PC (Intel Pentium III 1200 MMX, Intel, Santa Clara, CA, USA). This optical system was associated with a digitizing pad (Matrix Vision GmbH, Oppenweiler, Germany) and a histometry software package with image capturing capabilities (Image-Pro Plus, Media Cybernetics Inc., Immagini e Computer Snc, Milano, Italy).
2
Materials and methods
2.1
Surgical procedure
Skeletally mature (9 month old, 3.5 kg weigh) pathogen free (SPF) and virus antibody free (VAF) male New Zealand white rabbits (Crl:KBL(NZW)) obtained from Charles River Laboratories (Lieu-dit Oncins, France) were used. The protocol conformed to the guiding principles of ISO 10993-2 (Animal welfare requirements) and ISO 10993-1 (Part 6: tests for local effects after implantation) and was approved by the Ethical Committee of University of Chieti-Pescara, Italy.
The rabbits were anesthetized with intramuscular injections of fluanizone (0.7 mg/kg body weight) and diazepam (1.5 mg/kg body weight), and local anaesthesia was given using 1 mL of 2% lidocain/adrenalin solution. The medial surface of the tibiae was exposed via a skin excision with a periosteal flap . Care was taken to split the muscular layer by blunt dissection and to keep the periosteum intact apart from the longitudinal excision. Cylindrical noncritical-sized bone defects (2 mm in diameter) were performed in the medial diaphyseal face of rabbit tibia using a 2 mm diameter drill working at 300 rpm under constant copious saline irrigation. The bone excision was performed through the entire cortical thickness in order to reach the medullary space. Defects were filled with Biodentine (Septodont, Saint-Maur-des-Fossés, France; batch number B01767), MTA Plus with gel (Prevest Detpro Limited, Jammu, India; lot n. 41001) or ProRoot MTA (Dentsply Tulsa, Johnson City, TN, USA; batch number 09003850) as graft materials (n = 6 for each material in 6 different animals, following ISO 10993-1) . All cements were prepared following the manufacturer directions and inserted manually into the bone defects using a stainless steel spatula, and extruded into the medullary area. In the control group the noncritical-sized defects were left empty and were allowed to heal spontaneously.
The overlying soft tissues, periostium and fascia, were sutured with catgut and the skin with silk suture. Eight bone defects were created in each animal, 4 in the right tibia, and 4 in the left tibia ( Fig. 1 ).
Oxytetracycline dihydrate (Terramicina long Acting by Pfizer Italia srl) 100 mg/kg single dose and analgesics with tramadol hydrochloride (Altadol Abiogen Pharma S.p.A Italia) were given for 1 week. Sutures were removed 2 weeks after surgery. Postsurgical visits were scheduled daily to check the course of healing. No complications or deaths occurred in the postoperative period.
The animals were pharmacologically euthanized 30 days after surgery (following ISO 10993-1 for prolonged-permanent long-term contact of implant devices for bone tissue) with an overdose of intravenous Tanax (Intervet Italia srl, Peschiera Borromeo, Mi, Italy) under general anaesthesia with intramuscular injections of fluanizone (0.7 mg/kg body weight) and diazepam (1.5 mg/kg body weight). Then, the bone samples were retrieved and processed for histological analysis.
2.2
Histological specimen processing
The retrieved specimens were immediately stored in 10% buffered formalin and processed to obtain thin ground sections with the Precise 1 Automated System (Assing, Rome, Italy) . They were dehydrated in an ascending series of alcohol rinses and embedded in a glycolmethacrylate resin (Technovit 7200, VLC, Kulzer, Wehrheim, Germany). After polymerization, the specimens were sectioned longitudinally along the major axis of the rabbit tibiae with a high-precision diamond disc at about 150 μm and ground down to about 30 μm. Then, the slides were stained with acid fuchsin and toluidine blue.
2.3
Histological analysis
2.3.1
EDX microchemical analysis and ESEM examination
The histological sections were examined using an environmental scanning electron microscope (ESEM, Zeiss EVO 50; Carl Zeiss, Oberkochen, Germany) connected to a secondary electron detector for energy dispersive X-ray analysis (EDX; Oxford INCA 350 EDS, Abingdon, UK) using computer-controlled software (Inca Energy Version 18). The sections were examined uncoated at low vacuum (100 Pascal), 20 kV accelerating voltage, 8.5 mm working distance, 0.5 wt% detection level, 133 eV resolution, 100 microseconds amplification time, measuring time: 600 s for element mapping and 60 s for spectra. The resulting electron beam penetration inside was approx. 2 μm.
ESEM histological observations were performed at 100×, 1500× and 3000× magnification.
EDX microchemical analysis (elemental X-ray microanalysis) was carried out at random in areas of approx. 50 × 50 μm to evaluate the relative element content. Microanalysis (weight % and atomic %) with ZAF correction method was performed in full frame and spot mode to analyze entire areas or specific points respectively. The Ca/P, Ca/N and P/N ratios were calculated from the data to evaluate the degree of mineralization of the newly formed bone.
EDX element mapping was performed at the bone-material interface to detect the element distribution within the mature and neoformed bone and the presence in the surrounding tissues of elements from the implanted material.
Element mapping was performed using 512 × 384 pixel matrix, 30–40 frames, 100 μs dwelling time, 600–700 total reading time.
Line profile (line scans) through the bone-interface-material were performed to detect the variation content of Ca, P, S, C and N from the mature pre-existing bone towards the implanted material.
Scan line was carried out using 300 s reading time.
2.3.2
OM examination and histomorphometry
Histological analysis and histomorphometry of the percentage of newly formed bone, marrow spaces and residual biomaterial was carried out using a light microscope (Leitz Laborlux, Wetzlar, Germany) connected to a high resolution video camera (3CCD, JVC KY-F55B, JVC, Yokohama, Japan) and interfaced to a monitor and PC (Intel Pentium III 1200 MMX, Intel, Santa Clara, CA, USA). This optical system was associated with a digitizing pad (Matrix Vision GmbH, Oppenweiler, Germany) and a histometry software package with image capturing capabilities (Image-Pro Plus, Media Cybernetics Inc., Immagini e Computer Snc, Milano, Italy).
3
Results
3.1
ProRoot MTA
The histological section observed by ESEM ( Fig. 2 ) showed the surgical defect completely filled by compact material. Neoformed bone juxtaposed and strictly in contact with the material on its medullary side demonstrated the osteoinductive properties of ProRoot MTA.
EDX microchemical characterization ( Fig. 2 ) on the material displayed its constitutive elements i.e., Ca, Si, Bi and traces of Al.
Pre-existing trabecular bone showed Ca and P. Neoformed bone revealed Ca, P, absence of Bi, and traces of Si deriving from the material.
The Ca/N and P/N ratios were markedly higher in the pre-existing mature bone that in the newly formed bone, in particular in the new trabeculae adjacent to the material (likely the newest bone).
The Ca/P ratio in neoformed trabeculae was quite high, approaching the value of the mature bone, likely due to a more mature bone and/or to a diffusion of Ca (similarly to Si) to the surrounding/adjacent tissues. In addition, the high Ca/P ratio calculated in some interface analysis (see points 12 and 15) can be affected and raised by the constitutive Ca of the filling material.
The analysis of the interface at high magnification ( Fig. 3 ) displayed the perfect contact and continuity of the neoformed trabeculae with the material and well displayed the absence of gaps. No Bi or Al was detected in neoformed bone but only at the interface.
EDX ( Fig. 4 ) mapping showed the material elements, mainly Ca and Si, that clearly mark the filled area. Amounts of Al and sparse Bi charged particles were visible into the cement.
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