3D-printed MgO nanoparticle loaded polycaprolactone ß-tricalcium phosphate composite scaffold for bone tissue engineering applications: In-vitro and in-vivo evaluation.

Magnesium (Mg) plays an important role in controlling bone apatite structure and density and is a potential bioactive material in repairing critical-sized bone defects. In this study, we aimed to evaluate the effect of adding NanoMgO to polycaprolactone/beta-tricalcium phosphate (PCL/ß-TCP) scaffolds on bone regeneration. Novel 3D-printed porous PCL/ß-TCP composite scaffolds containing 10% nanoMgO were fabricated by fused deposition modeling (FDM) and compared with PCL/ß-TCP (1:1) scaffolds (control). The morphology and physicochemical properties of the scaffolds were characterized by ATR-FTIR, XRD, scanning electron microscope-energy dispersive X-ray analysis (SEM-EDX), transmission-electron-microscopy (TEM), water contact angle, and compressive strength tests and correlated to its cytocompatibility and osteogenic capacity in-vitro. To evaluate in-vivo osteogenic capacity, bone-marrow-derived stem cell (BMSC)-loaded scaffolds were implanted into 8 mm rat critical-sized calvarial defects for 12 weeks. The hydrophilic scaffolds showed 50% porosity (pore size = 504 µm). MgO nanoparticles (91.5 ± 27.6 nm) were homogenously dispersed and did not adversely affect BMSCs' viability and differentiation. Magnesium significantly increased elastic modulus, pH, and degradation. New bone formation (NBF) in Micro-CT was 30.16 ± 0.31% and 23.56 ± 1.76% in PCL/ß-TCP/nanoMgO scaffolds with and without BMSCs respectively, and 19.38 ± 2.15% and 15.75 ± 2.24% in PCL/ß-TCP scaffolds with and without BMSCs respectively. Angiogenesis was least remarkable in PCL/ß-TCP compared with other groups (p < .05). Our results suggest that the PCL/ß-TCP/nanoMgO scaffold is a more suitable bone substitute compared to PCL/ß-TCP in critical-sized calvarial defects.

In vivo evaluation of bone regeneration behavior of novel ß-tricalcium phosphate/layered double hydroxide nanocomposite granule as bone graft substitutes.

This study was based on in vivo assessment of bone regeneration capacity of synthesized porous ß-tricalcium phosphate (ß-TCP) nanocomposite granules and aimed to explore the effects of fabricated ß-TCP granules reinforced with layered double hydroxides (LDH) nanoclay compared to ß-TCP granules, in terms of osteoconductivity and biodegradability. Granules with diameters of 2-3 mm were implanted into cavities drilled in rabbit distal femur and were left in situ for up to 3 months. The mechanical study demonstrated that the presence of LDH nanoparticles in ß-TCP granules resulted in a significant increase in compressive modulus from 174.4 to 231.4 MPa, while the porosity was constant at 76%-80%. The results revealed that the obtained granules showed no cytotoxicity. In this study, x-ray radiographic, micro-computed tomography, and histological staining analysis were taken to evaluate the percentage of bone ingrowth and biodegradability of the porous granules. The results exhibited that both granules support bone regeneration and also the amount of new bone formation in the bone defect filled with both granules was almost six times higher than the empty defects. Although no significant difference in bone formation for two different granules was observed, a higher biodegradability was detected in ß-TCP granules in comparison to ß-TCP/LDH granules. Overall, the addition of LDH nanoclay (10%) enhanced the physicochemical and mechanical properties of ß-TCP granules while it is biological and osteoconductity properties have been maintained and its biodegradation rate has been decreased.

The correlation between osseointegration and bonding strength at the bone-implant interface: In-vivo & ex-vivo investigations on hydroxyapatite and hydroxyapatite/titanium coatings.

This study investigated the effects of hydroxyapatite (HA) and hydroxyapatite/titanium (HA/Ti) coatings on osseointegration and bonding strength at the bone-implant interface. The coatings were made using air plasma spray (APS), and three study groups were examined: 1) Uncoated commercial pure titanium (CP-Ti) rods; 2) HA-coated CP-Ti rods, and 3) Composite of 50 %wt HA + 50 %wt Ti coated CP-Ti rods. The rods were implanted into the distal femurs and proximal tibias of fifteen New Zealand white rabbits, and 8 weeks after the implantation, the samples were harvested. The results of pull-out tests showed that the ultimate strength of HA and HA/Ti coatings were significantly greater than the uncoated samples (P < 0.05). Moreover, even though the histological evaluations showed significantly greater osseointegration of HA/Ti composite coatings compared with HA coatings (P < 0.05), nonetheless, the composite of HA/Ti offers no significant increase in the ultimate strength, stiffness, and bonding strength at the bone-implant interface, compared with the HA group (P > 0.05). Thus, in an eight-week study, there was no linear correlation between the osseointegration and the bonding strength at the bone-implant interface. The results of this work may imply that the extent of osseointegration at the bone-implant interface does not necessarily determine the value of the bonding strength at the bone-implant interface. It is speculated that, in a longer-term study, a greater quality of bone formation may occur during osseointegration, between the implant and its adjacent bone, which can lead to a more enhanced bonding strength, compared with the 8-weeks post-surgery follow up.

Direct pulp capping with autologous bone marrow derived stem cells in dogs.

Bone-marrow derived stem cells (BMSCs) can differentiate into several mesenchymal cell lines that are suitable for bone and dental tissue engineering. This study was aimed to assess the efficacy of cell therapy in direct pulp capping (DPC) of canine teeth using autologous BMSCs along with collagen/hydroxyapatite hybrid scaffold in terms of the quantity and quality of calcified bridge formation. The teeth were randomly divided into three groups of DPC with mineral trioxide aggregate (MTA), hydroxyapatite/collagen hybrid scaffold alone and BMSCs with hydroxyapatite/collagen hybrid scaffold. DPC was performed under general anesthesia in cavities prepared on the buccal surfaces of mandibular and maxillary premolars of the same dogs from which, stem cells had been isolated. All cavities were then restored with light-cure resin modified glass ionomer cement. Histomorphometric assessments after 12 weeks showed formation of dentinal bridge following DPC with BMSCs and MTA. The efficacy of MTA for calcified bridge formation following DPC was significantly higher than that of BMSCs plus hybrid scaffold. According to the present study, we concluded DPC using BMSCs and hybrid scaffold did not provide clinically noticeable results in canine patients.

Prefabrication technique by preserving a muscular pedicle from masseter muscle as an in vivo bioreactor for reconstruction of mandibular critical-sized bone defects in canine models.

In vivo bioreactors serve as regenerative niches that improve vascularization and regeneration of bone grafts. This study has evaluated the masseter muscle as a natural bioreactor for ßTCP or PCL/ßTCP scaffolds, in terms of bone regeneration. The effect of pedicle preservation, along with sole, or MSC- or rhBMP2-combined application of scaffolds, has also been studied. Twenty-four mongrel dogs were randomly placed in six groups, including ßTCP, ßTCP/rhBMP2, ßTCP/MSCs, PCL/ßTCP, PCL/ßTCP/rhBMP2, and PCL/ßTCP/MSCs. During the first surgery, the scaffolds were implanted into the masseter muscle for being prefabricated. After 2 months, each group was divided into two subgroups prior to mandibular bone defect reconstruction; one with a preserved vascularized pedicle and one without. After 12 weeks, animals were euthanized, and new bone formation was evaluated using histological analysis. Histological analysis showed that all ß-TCP scaffold groups had resulted in significantly greater rates of new bone formation, either with a pedicle surgical approach or non-pedicle surgical approach, comparing to their parallel groups of ßTCP/PCL scaffolds (p ≤ .05). Pedicled ß-TCP scaffold groups that were treated with either rhBMP2 (48.443% ± 0.250%) or MSCs (46.577% ± 0.601%) demonstrated the highest rates of new bone formation (p ≤ .05). Therefore, masseter muscle can be used as a local in vivo bioreactor with potential clinical advantages in reconstruction of human mandibular defects. In addition, scaffold composition, pedicle preservation, and treatment with MSCs or rhBMP2, influence new bone formation and scaffold degradation rates in the prefabrication technique.

Comparison of engineered cartilage based on BMSCs and chondrocytes seeded on PVA-PPU scaffold in a sheep model.

In this study, polyvinyl alcohol hydrogel chains were crosslinked by polyurethane in order to synthesize a suitable substrate for cartilage lesions. The substrate was fully characterized, and in vitro and in vivo investigations were conducted based on a sheep model. In vitro tests were performed based on the chondrocyte cells with the Alcian Blue and safranin O staining in order to prove the presence of proteoglycan on the surface of the synthesized substrate, which has been secreted by cultures of chondrocytes. Furthermore, the expression of collagen type I, collagen type II, aggrecan, and Sox9 was presented in the chondrocyte cultures on the synthesized substrate through RT-PCR. In addition, the H&E analysis and other related tests demonstrated the formation of neocartilage tissue in a sheep model. The results were found to be promising for cartilage tissue engineering and verified that the isolated chondrocyte cultures on the synthesized substrate retain their original composition.

Dental implants' stability dependence on rotational speed and feed-rate of drilling: In-vivo and ex-vivo investigations.

This study aimed to explore the effects of drilling rotational speed and feed-rate on the stability of dental implants through in-vivo and ex-vivo experiments. To this end, a total of 16 identical dental implants were inserted in the mandible of four dogs. The osteotomies were made with two drilling rotational speeds, i.e., 800 and 1500 rpm, and two different feed-rates, i.e., 1 and 2 mm/s. Implant stability quotients (ISQs) were recorded immediately after inserting implants and then each week for four subsequent weeks. Then, all animals were euthanized, and a bone sample containing the implants was extracted from each hemi-mandible for the pull-out test. A two-way ANOVA was performed for ISQs, and pull-out strengths (PoS), and the significance level was set to <0.05. The effect of rotational speed and feed-rate, used in this study, on the primary stability quotients was not significant (P > 0.05). Increasing the rotational speed from 800 to 1500 rpm significantly increased both ISQ and PoS values at the end of the 4th week after the implantation (P = 0.022 and P = 0.001, respectively). Moreover, by decreasing the feed-rate from 2 to 1 mm/s, a significant increase in PoSs of the dental implants was observed four weeks after the implantation (P = 0.019). Results of this study showed that either by increasing drilling rotational speed, here from 800 to 1500 rpm, or by reducing feed-rate, here from 2 to 1 mm/s, the secondary stability would be reinforced. Further investigations are needed to see if and how the conclusions made in this study can be generalized.

Linear Momenta Transferred to the Dental Implant-Bone and Natural Tooth-PDL-Bone Constructs Under Impact Loading: A Comparative in-vitro and in-silico Study.

During dental trauma, periodontal ligament (PDL) contributes to the stability of the tooth-PDL-bone structure. When a dental implant is inserted into the bone, the dental implant-bone construct will be more prone to mechanical damage, caused by impact loading, than the tooth-PDL-bone construct. In spite of the prevalence of such traumas, the behavioral differences between these two constructs have not been well-understood yet. The main goal of this study was to compare the momentum transferred to the tooth-PDL-bone and dental implant-bone constructs under impact loading. First, mechanical impact tests were performed on six canine mandibles of intact (N = 3) and implanted (N = 3) specimens using a custom-made drop tower apparatus, from release heights of 1, 2, and 3 cm. Next, computed tomography-based finite element models were developed for both constructs, and the transferred momenta were calculated. The experimental results indicated that, for the release heights of 1, 2, and 3 cm, the linear momenta transferred to the dental implant-bone construct were 33.1, 31.0, and 27.5% greater than those of the tooth-PDL-bone construct, respectively. Moreover, results of finite element simulations were in agreement with those of the experimental tests (error <7.5%). This work tried to elucidate the effects of impact loading on the dental implant-bone and tooth-PDL-bone constructs using both in-vitro tests and validated in-silico simulations. The findings can be employed to modify design of the current generation of dental implants, based on the lessons one can take from the biomechanical behavior of a natural tooth structure.

Recovery of inferior alveolar nerve by photobiomodulation therapy using two laser wavelengths: A behavioral and immunological study in rat.

Postoperative sensory disturbances of inferior alveolar nerve (IAN) are major challenges in dental procedures. We aimed to investigate the effect of photobiomodulation therapy (PBMT) with 810 nm and 980 nm diode lasers on behavioral and immunological factors in a rat IAN crush model. Seventy-two rats were randomly assigned to the four groups of 810 nm laser (crush injury+810 nm laser; 6 J/cm2, 15 sessions, every 48 h), 980 nm laser (crush injury+980 nm laser; same protocol), control (crush injury without irradiation), and sham surgery (no crush injury and no irradiation). The neurosensory response of IAN was evaluated by Von Frey behavioral test before (baseline) and post-surgery in a period of one month. Changes of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), nuclear factor-kappa B (NF-κB), TNF-α, and IL-1ß, were assessed on days 2 and 30 post injury. Data were analyzed for significant differences by repeated measures and one-way ANOVA (p < .05). One day after surgery, all rats subjected to nerve injury showed significant increase in the withdrawal threshold of von Frey test compared to the baseline (p = .02 for control and p = .03 for laser groups). The threshold gradually returned to the baseline scores in 810 nm, 980 nm, and control groups from days 11, 17, and 29, respectively. There was a significant lower withdrawal threshold in 810 nm and 980 nm laser groups compared to the control group in days 11 to 19 and 9 to 23, respectively. At both time points, the levels of NGF and BDNF were significantly higher in 810 nm laser group compared to the control group. There was a significant difference between laser and control groups regarding NF-κB expression (all p values<.001). TNF-α and IL-1ß were significantly lower in laser groups compared to the control group (all p values < .001). PBMT with 810 and 980 nm diode laser protocol used in this study, promoted the neurosensory recovery of IAN after crush injury in rats. In addition, application of 810 nm diode laser was associated with more improvement in immunological responses compared to that of 980 nm laser.

The effects of bone implants' coating mechanical properties on osseointegration: In vivo, in vitro, and histological investigations.

The main goal of this work was to investigate the effects of implants coatings' mechanical properties and morphology on the osseointegration. In order to produce different mechanical properties of coatings, two thermal spray techniques, high velocity oxy-fuel (HVOF) and air plasma spray (APS) were employed. Titanium pins were coated and implanted into the distal femurs and proximal tibias of fifteen New Zealand white rabbits, equally distributed in three study groups, and a total of 20 pins implanted in each group. Eight weeks after insertion, the rabbits were euthanized and the femur samples were taken out for biomechanical tests and tibia samples for histological evaluations of osseointegration. Scanning electron microscopy results showed enhanced density and a better morphology of HVOF coatings, compared to APS samples, and X-ray diffraction characterized an enhanced crystallinity of HVOF coatings. Nanoindentation tests revealed greater hardness and elastic modulus of HVOF coatings, whereas greater tensile residual stress and more pronounced creep was observed for APS coatings. Neither in biomechanical tests, nor in the histological analyses, a significant difference was observed between HVOF and APS coated samples (p > 0.05, and p > 0.05, respectively). The lack of significant difference between the HVOF and APS coated implants' osseointegration rejected our hypothesis to have a more enhanced osseointegration due to a better morphology, as well as stronger mechanical properties of HA coatings. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 2679-2691, 2018.