On-Growth and In-Growth Osseointegration Enhancement in PM Porous Ti-Scaffolds by Two Different Bioactivation Strategies: Alkali Thermochemical Treatment and RGD Peptide Coating.

A lack of primary stability and osteointegration in metallic implants may result in implant loosening and failure. Adding porosity to metallic implants reduces the stress shielding effect and improves implant performance, allowing the surrounding bone tissue to grow into the scaffold. However, a bioactive surface is needed to stimulate implant osteointegration and improve mechanical stability. In this study, porous titanium implants were produced via powder sintering to create different porous diameters and open interconnectivity. Two strategies were used to generate a bioactive surface on the metallic foams: (1) an inorganic alkali thermochemical treatment, (2) grafting a cell adhesive tripeptide (RGD). RGD peptides exhibit an affinity for integrins expressed by osteoblasts, and have been reported to improve osteoblast adhesion, whereas the thermochemical treatment is known to improve titanium implant osseointegration upon implantation. Bioactivated scaffolds and control samples were implanted into the tibiae of rabbits to analyze the effect of these two strategies in vivo regarding bone tissue regeneration through interconnected porosity. Histomorphometric evaluation was performed at 4 and 12 weeks after implantation. Bone-to-implant contact (BIC) and bone in-growth and on-growth were evaluated in different regions of interest (ROIs) inside and outside the implant. The results of this study show that after a long-term postoperative period, the RGD-coated samples presented higher quantification values of quantified newly formed bone tissue in the implant's outer area. However, the total analyzed bone in-growth was observed to be slightly greater in the scaffolds treated with alkali thermochemical treatment. These results suggest that both strategies contribute to enhancing porous metallic implant stability and osteointegration, and a combination of both strategies might be worth pursuing.

Computed tomography and histological evaluation of xenogenic and biomimetic bone grafts in three-wall alveolar defects in minipigs.

OBJECTIVES: This study aimed to compare the performance of a xenograft (XG) and a biomimetic synthetic graft (SG) in three-wall alveolar defects in minipigs by means of 3D computerised tomography and histology. MATERIALS AND METHODS: Eight minipigs were used. A total of eight defects were created in the jaw of each animal, three of which were grafted with XGs, three with SGs, and two were left empty as a negative control. The allocation of the different grafts was randomised. Four animals were euthanised at 6 weeks and four at 12 weeks. The grafted volume was then measured by spiral computed tomography to assess volume preservation. Additionally, a histological analysis was performed in undecalcified samples by backscattered scanning electron microscopy and optical microscopy after Masson's trichrome staining. RESULTS: A linear mixed-effects model was applied considering four fixed factors (bone graft type, regeneration time, anatomic position, and maxilla/mandible) and one random factor (animal). The SG exhibited significantly larger grafted volume (19%) than the XG. The anterior sites preserved better the grafted volume than the posterior ones. Finally, regeneration time had a positive effect on the grafted volume. Histological observations revealed excellent osseointegration and osteoconductive properties for both biomaterials. Some concavities found in the spheroidal morphologies of SGs were associated with osteoclastic resorption. CONCLUSIONS: Both biomaterials met the requirements for bone grafting, i.e. biocompatibility, osseointegration, and osteoconduction. Granule morphology was identified as an important factor to ensure a good volume preservation. CLINICAL RELEVANCE: Whereas both biomaterials showed excellent osteoconduction, SGs resulted in better volume preservation.

COVID-19: The immediate response of european academic dental institutions and future implications for dental education.

The COVID-19 pandemic has had an immediate and dramatic impact on dental education. The Association of Dental Education in Europe decided to carry out an investigation to assess the immediate response of European Academic Dental Institutions. An online survey was sent to both member and non-member dental schools to investigate the impact on non-clinical and clinical education, assessment and the well-being/pastoral care measures implemented. The preliminary findings and discussion are presented in this paper, for the responses collected between the 25 March and 5 April 2020. The survey at this time of publication is ongoing, and detailed results can be accessed https://adee.org/covid-19-european-dental-education%E2%80%99s-immediate-response.

A core curriculum in the biological and biomedical sciences for dentistry.

INTRODUCTION: The biomedical sciences (BMS) are a central part of the dental curriculum that underpins teaching and clinical practice in all areas of dentistry. Although some specialist groups have proposed curricula in their particular topic areas, there is currently no overarching view of what should be included in a BMS curriculum for undergraduate dental programmes. To address this, the Association for Dental Education in Europe (ADEE) convened a Special Interest Group (SIG) with representatives from across Europe to develop a consensus BMS curriculum for dental programmes. CURRICULUM: This paper summarises the outcome of the deliberations of this SIG and details a consensus view from the SIG of what a BMS curriculum should include. CONCLUSIONS: Given the broad nature of BMS applied to dentistry, this curriculum framework is advisory and seeks to provide programme planners with an indicative list of topics which can be mapped to specific learning objectives within their own curricula. As dentistry becomes increasingly specialised, these will change, or some elements of the undergraduate curriculum may move to the post-graduate setting. So, this document should be seen as a beginning and it will need regular review as BMS curricula in dentistry evolve.

Elements of morphology: Standard terminology for the teeth and classifying genetic dental disorders.

Dental anomalies occur frequently in a number of genetic disorders and act as major signs in diagnosing these disorders. We present definitions of the most common dental signs and propose a classification usable as a diagnostic tool by dentists, clinical geneticists, and other health care providers. The definitions are part of the series Elements of Morphology and have been established after careful discussions within an international group of experienced dentists and geneticists. The classification system was elaborated in the French collaborative network "TÊTECOU" and the affiliated O-Rares reference/competence centers. The classification includes isolated and syndromic disorders with oral and dental anomalies, to which causative genes and main extraoral signs and symptoms are added. A systematic literature analysis yielded 408 entities of which a causal gene has been identified in 79%. We classified dental disorders in eight groups: dental agenesis, supernumerary teeth, dental size and/or shape, enamel, dentin, dental eruption, periodontal and gingival, and tumor-like anomalies. We aim the classification to act as a shared reference for clinical and epidemiological studies. We welcome critical evaluations of the definitions and classification and will regularly update the classification for newly recognized conditions.

Vascularized Periosteal Flaps Accelerate Osteointegration and Revascularization of Allografts in Rats.

BACKGROUND: Surgical reconstruction of large bone defects with structural bone allografts can restore bone stock but is associated with complications such as nonunion, fracture, and infection. Vascularized reconstructive techniques may provide an alternative in the repair of critical bone defects; however, no studies specifically addressing the role of vascularized periosteal flaps in stimulating bone allograft revascularization and osseointegration have been reported. QUESTIONS/PURPOSES: (1) Does a vascularized periosteal flap increase the likelihood of union at the allograft-host junction in a critical-size defect femoral model in rats? (2) Does a vascularized periosteal flap promote revascularization of a critical-size defect structural bone allograft in a rat model? (3) What type of ossification occurs in connection with a vascularized periosteal flap? METHODS: Sixty-four rats were assigned to two equal groups. In both the control and experimental groups, a 5-cm critical size femoral defect was created in the left femur and then reconstructed with a cryopreserved structural bone allograft and intramedullary nail. In the experimental group, a vascularized periosteal flap from the medial femoral condyle, with a pedicle based on the descending genicular vessels, was associated with the allograft. The 32 rats of each group were divided into subgroups of 4-week (eight rats), 6-week (eight rats), and 10-week (16 rats) followup. At the end of their assigned followup periods, the animals were euthanized and their femurs were harvested for semiquantitative and quantitative analysis using micro-CT (all followup groups), quantitative biomechanical evaluation (eight rats from each 10-week followup group), qualitative confocal microscopic, backscattered electron microscopic, and histology analysis (4-week and 6-week groups and eight rats from each 10-week followup group). When making their analyses, all the examiners were blinded to the treatment groups from which the samples came. RESULTS: There was an improvement in allograft-host bone union in the 10-week experimental group (odds ratio [OR], 19.29 [3.63-184.50], p < 0.05). In contrast to control specimens, greater bone neoformation in the allograft segment was observed in the experimental group (OR [4-week] 63.3 [39.6-87.0], p < 0.05; OR [6-week] 43.4 [20.5-66.3], p < 0.05; OR [10-week] 62.9 [40.1-85.7], p < 0.05). In our biomechanical testing, control samples were not evaluable as a result of premature breakage during the embedding and assembly processes. Therefore, experimental samples were compared with untreated contralateral femurs. No difference in torsion resistance pattern was observed between both groups. Both backscattered electron microscopy and histology showed newly formed bone tissue and osteoclast lacunae, indicating a regulated process of bone regeneration of the initial allograft in evaluated samples from the experimental group. They also showed intramembranous ossification produced by the vascularized periosteal flap in evaluated samples from the experimental group, whereas samples from the control group showed an attempted endochondral ossification in the allograft-host bone junctions. CONCLUSIONS: A vascularized periosteal flap promotes and accelerates allograft-host bone union and revascularization of cryopreserved structural bone allografts through intramembranous ossification in a preclinical rat model. CLINICAL RELEVANCE: If large-animal models substantiate the findings made here, this approach might be used in allograft reconstructions for critical defects using fibular or tibial periosteal flaps as previously described.

A new standardized-automatic method for bone-to-implant contact histomorphometric analysis based on backscattered scanning electron microscopy images.

AIM: To establish an image analysis procedure for measuring the bone-to-implant contact (BIC) by a systematic non-subjective approach based on backscattered scanning electron microscopy (BS-SEM) images. MATERIAL AND METHODS: A total of 36 dental implants (9 mm length, Ø 4.0 mm with a SBM surface) were implanted in six beagle dog mandibles. The implants were removed after 1, 2, 4, 6, and 8 weeks and then embedded in resin and cut along their long axis. Sample observation was performed by BS-SEM, acquiring 10 to 16 images per sample. Image processing and BIC determination were performed using the Fiji image processing package. Images were stitched, filtered, and thresholded to obtain a binary image of the whole implant that finally was dilated and outlined. The length of this outline was measured as the maximum possible BIC. The regions of coincidence between this line and the bone were measured as the real BIC. RESULTS: The proposed methodology for BIC determination, based on SEM, which has a much higher resolution than optical microscopy, allows the acquisition of highly discriminative images with great contrast between implant and bone. The high resolution and high contrast in SEM images provide more accurate results than those obtained by classical methods. Furthermore, the methodology of image analysis described in this study delineates precisely and automatically the contour of the implant, which results in non-biased measurements. The average percentage of BIC was 35%, ranging from 24.7 to 45.5%. These values were similar to the results documented in the literature for implants of similar roughness in animal models. CONCLUSIONS: A novel, non-subjective, and systematic method for measuring BIC is described based on BS-SEM images. The proposed methodology minimizes the shortcomings of the results obtained by previously described methods.

Poly-3-hydroxybutyrate-silver nanoparticles membranes as advanced antibiofilm strategies for combatting peri-implantitis.

Dental implant success is threatened by peri-implantitis, an inflammation leading to implant failure. Conventional treatments struggle with the intricate microbial and host factors involved. Antibacterial membranes, acting as barriers and delivering antimicrobials, may offer a promising solution. Thus, this study highlights the potential of developing antibacterial membranes of poly-3-hydroxybutyrate and silver nanoparticles (Ag Nps) to address peri-implantitis challenges, discussing design and efficacy against potential pathogens. Electrospun membranes composed of PHB microfibers and Ag Nps were synthesized in a blend of DMF/chloroform at three different concentrations. Various studies were conducted on the characterization and antimicrobial activity of the membranes. The synthesized Ag Nps ranged from 4 to 8 nm in size. Furthermore, Young's modulus decreased, reducing from 13.308 MPa in PHB membranes without Ag Nps to 0.983 MPa in PHB membranes containing higher concentrations of Ag Nps. This demonstrates that adding Ag Nps results in a less stiff membrane. An increase in elongation at break was noted with the rise in Ag Nps concentration, from 23.597 % in PHB membranes to 60.136 % in PHB membranes loaded with Ag Nps. The antibiotic and antibiofilm activity of the membranes were evaluated against Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus mutans, and Candida albicans. The results indicated that all PHB membranes containing Ag Nps exhibited potent antibacterial activity by inhibiting the growth of biofilms and planktonic bacteria. However, inhibition of C. albicans occurred only with the PHB-Ag Nps C membrane. These findings emphasize the versatility and potential of Ag Nps-incorporated membranes as a multifunctional approach for preventing and addressing microbial infections associated with peri-implantitis. The combination of antibacterial and antibiofilm properties in these membranes holds promise for improving the management and treatment of peri-implantitis-related complications.

3D printing with star-shaped strands: A new approach to enhance in vivo bone regeneration.

Concave surfaces have shown to promote bone regeneration in vivo. However, bone scaffolds obtained by direct ink writing, one of the most promising approaches for the fabrication of personalized bone grafts, consist mostly of convex surfaces, since they are obtained by microextrusion of cylindrical strands. By modifying the geometry of the nozzle, it is possible to print 3D structures composed of non-cylindrical strands and favor the presence of concave surfaces. In this work, we compare the in vivo performance of 3D-printed calcium phosphate scaffolds with either conventional cylindrical strands or star-shaped strands, in a rabbit femoral condyle model. Monocortical defects, drilled in contralateral positions, are randomly grafted with the two scaffold configurations, with identical composition. The samples are explanted eight weeks post-surgery and assessed by µ-CT and resin-embedded histological observations. The results reveal that the scaffolds containing star-shaped strands have better osteoconductive properties, guiding the newly formed bone faster towards the core of the scaffolds, and enhance bone regeneration, although the increase is not statistically significant (p > 0.05). This new approach represents a turning point towards the optimization of pore shape in 3D-printed bone grafts, further boosting the possibilities that direct ink writing technology offers for patient-specific applications.

Perioral Aerosol Sequestration Suction Device Effectively Reduces Biological Cross-Contamination in Dental Procedures.

OBJECTIVE: The infection risk during dental procedures is a common concern for dental professionals which has increased due to coronavirus (severe acute respiratory syndrome coronavirus 2) pandemic. The development of devices to specifically mitigate cross-contamination by droplet/splatter is crucial to stop infection transmission. The objective of this study is to assess the effectiveness of a perioral suction device (Oral BioFilter, OBF) to reduce biological contamination spread during dental procedures. MATERIALS AND METHODS: Forty patients were randomized 1:1 to a standard professional dental hygiene treatment with OBF and without. Adenosine triphosphate (ATP) bioluminescence assay was used to evaluate the spread of potential contaminants. The total number of relative light units (RLU) from key dental operatory locations: operator's face-shield, back of the surgical operator's-gloves, patient's safety-goggles, and instrumental table were measured. Percentage contamination reductions between control and OBF were compared. STATISTICAL ANALYSIS: Primary outcome, total RLU, was analyzed by comparing the means of logged data, using a two-sided two-sample t-test. Secondary outcomes as RLU of logged data for the different locations were analyzed in the same way. Proportion of patients from whom different locations reported events (clean, acceptable, and failure) were analyzed by using Fisher's exact test. RESULTS: For the whole dental environment, RLUs reduction (<150 units) achieved with OBF was 98.4% (97.4-99%). By dental operatory location the reduction in RLUs was from 99.6%, on the operator face-shield, to 83% on instrumental table. The control group reported a very high percentage of failures, (>300) being 100% on the surfaces closer to the patient's mouth and decreasing to 70% on instrumental table. In contrast, the higher failure percentage in the OBF group was found on the patient's goggles (40%), while the operator face-shield showed an absence of contamination. CONCLUSION: OBF device has shown efficient reduction of biological aerosol cross-contamination during dental procedures as proved by ATP-bioluminescence assay. Nevertheless, for maximum safety, its use must be combined with standard protective gear such as goggles, face shield, and surgical gloves.

Nanostructured Zn-Substituted Monetite Based Material Induces Higher Bone Regeneration Than Anorganic Bovine Bone and ß-Tricalcium Phosphate in Vertical Augmentation Model in Rabbit Calvaria.

The capacity of a nanostructured multicomponent material composed of Zn-substituted monetite, amorphous calcium phosphate, hydroxyapatite and silica gel (MSi) to promote vertical bone augmentation was compared with anorganic bovine bone (ABB) and synthetic ß-tricalcium phosphate (ß-TCP). The relation between biological behavior and physicochemical properties of the materials was also studied. The in vivo study was conducted in a vertical bone augmentation model in rabbit calvaria for 10 weeks. Significant differences in the biological behavior of the materials were observed. MSi showed significantly higher bone regeneration (39%) than ABB and ß-TCP (24%). The filled cylinder volume was similar in MSi (92%) and ABB (91%) and significantly lower in ß-TCP (81%) implants. In addition, ß-TCP showed the highest amount of non-osteointegrated particles (17%). MSi was superior to the control materials because it maintains the volume of the defect almost full, with the highest bone formation, the lowest number of remaining particles, which are almost fully osteointegrated and having the lowest amount of connective tissue. Besides, the bone formed was mature, with broad trabeculae, high vascularization and osteogenic activity. MSi resorbs gradually over time with an evident increment of the porosity and simultaneous colonization for vascularized new bone. In addition, the osteoinductive behavior of MSi material was evidenced.

Hydrothermal processing of 3D-printed calcium phosphate scaffolds enhances bone formation in vivo: a comparison with biomimetic treatment.

Hydrothermal (H) processes accelerate the hydrolysis reaction of α-tricalcium phosphate (α-TCP) compared to the long-established biomimetic (B) treatments. They are of special interest for patient-specific 3D-printed bone graft substitutes, where the manufacturing time represents a critical constraint. Altering the reaction conditions has implications for the physicochemical properties of the reaction product. However, the impact of the changes produced by the hydrothermal reaction on the in vivo performance was hitherto unknown. The present study compares the bone regeneration potential of 3D-printed α-TCP scaffolds hardened using these two treatments in rabbit condyle monocortical defects. Although both consolidation processes resulted in biocompatible scaffolds with osseointegrative and osteoconductive properties, the amount of newly formed bone increased by one third in the hydrothermal vs the biomimetic samples. B and H scaffolds consisted mostly of high specific surface area calcium-deficient hydroxyapatite (38 and 27 m2 g-1, respectively), with H samples containing also 10 wt.% ß-tricalcium phosphate (ß-TCP). The shrinkage produced during the consolidation process was shown to be very small in both cases, below 3%, and smaller for H than for B samples. The differences in the in vivo performance were mainly attributed to the distinct crystallisation nanostructures, which proved to have a major impact on permeability and protein adsorption capacity, using BSA as a model protein, with B samples being highly impermeable. Given the crucial role that soluble proteins play in osteogenesis, this is proposed to be a relevant factor behind the distinct in vivo performances observed for the two materials. STATEMENT OF SIGNIFICANCE: The possibility to accelerate the consolidation of self-setting calcium phosphate inks through hydrothermal treatments has aroused great interest due to the associated advantages for the development of 3D-printed personalised bone scaffolds. Understanding the implications of this approach on the in vivo performance of the scaffolds is of paramount importance. This study compares, for the first time, this treatment to the long-established biomimetic setting strategy in terms of osteogenic potential in vivo in a rabbit model, and relates the results obtained to the physicochemical properties of the 3D-printed scaffolds (composition, crystallinity, nanostructure, nanoporosity) and their interaction with soluble proteins.

Effect of Allogeneic Cell-Based Tissue-Engineered Treatments in a Sheep Osteonecrosis Model.

Osteonecrosis of the femoral head (ONFH) is defined as a tissue disorder and successive subchondral bone collapse resulting from an ischemic process, which may progress to hip osteoarthritis. Cell therapy with multipotent bone marrow mesenchymal stromal cells (BM-MSC) of autologous origin appears to be safe and has shown regenerative potential in previous preclinical and clinical studies. The use of allogeneic cells is far more challenging, but may be a promising alternative to use of autologous cells. Moreover, an optimized dosage of cells from an allogeneic source is needed to obtain off-the-shelf tissue engineering products (TEPs). The purpose of this study was to evaluate the efficacy of a TEP composed of undifferentiated ex vivo expanded BM-MSC of allogeneic origin, combined with bone matrix particles in variable doses. A comparative analysis of TEP's bone regenerative properties against its autologous counterpart was performed in an early-stage ONFH preclinical model in mature sheep. Allogeneic BM-MSC groups demonstrated bone regeneration capacity in osteonecrotic lesions equivalent to autologous BM-MSC groups 6 weeks after treatment. Likewise, stimulation of bone regeneration by a low cell dose of 0.5 × 106 BM-MSC/cm3 was equivalent to that of a high cell dose, 5 × 106 BM-MSC/cm3. Neither local nor systemic immunological reactions nor tumorigenesis were reported, strengthening the safety profile of allogeneic BM-MSC therapy in this model. Our results suggest that low-dose allogeneic BM-MSC is sufficient to promote bone regeneration in femoral head osteonecrotic lesions, and should be considered in translation of new allogeneic cell-based TEPs to human clinics. Impact statement Cell therapy and tissue engineering hold promise as novel regenerative therapies for musculoskeletal diseases, and particularly in bone regeneration strategies. In this article, we report the evaluation of the efficacy of an allogeneic cell-based tissue engineering product (TEP) in an early-stage osteonecrosis of the femoral head preclinical model in skeletally mature sheep. Moreover, we demonstrate its bone regeneration capacity and safety in vivo and its equivalence to autologous counterparts. These findings have important implications for the translation of new allogeneic cell-based TEPs to human clinics.

Biofunctionalization with a TGFß-1 Inhibitor Peptide in the Osseointegration of Synthetic Bone Grafts: An In Vivo Study in Beagle Dogs.

OBJECTIVES: The aim of this research was to determine the osseointegration of two presentations of biphasic calcium phosphate (BCP) biomaterial-one untreated and another submitted to biofunctionalization with a TGF-ß1 inhibitor peptide, P144, on dental alveolus. Materials and Methods: A synthetic bone graft was used, namely, (i) Maxresorb® (Botiss Klockner) (n = 12), and (ii) Maxresorb® (Botiss Klockner) biofunctionalized with P144 peptide (n = 12). Both bone grafts were implanted in the two hemimandibles of six beagle dogs in the same surgical time, immediately after tooth extraction. Two dogs were sacrificed 2, 4, and 8 weeks post implant insertion, respectively. The samples were submitted to histomorphometrical and histological analyses. For each sample, we quantified the new bone growth and the new bone formed around the biomaterial's granules. After optical microscopic histological evaluation, selected samples were studied using backscattered scanning electron microscopy (BS-SEM). Results: The biofunctionalization of the biomaterial's granules maintains a stable membranous bone formation throughout the experiment timeline, benefitting from the constant presence of vascular structures in the alveolar space, in a more active manner that in the control samples. Better results in the experimental groups were proven both by quantitative and qualitative analysis. Conclusions: Synthetic bone graft biofunctionalization results in slightly better quantitative parameters of the implant's osseointegration. The qualitative histological and ultramicroscopic analysis shows that biofunctionalization may shorten the healing period of dental biomaterials.

Impact of Biomimicry in the Design of Osteoinductive Bone Substitutes: Nanoscale Matters.

Bone apatite consists of carbonated calcium-deficient hydroxyapatite (CDHA) nanocrystals. Biomimetic routes allow fabricating synthetic bone grafts that mimic biological apatite. In this work, we explored the role of two distinctive features of biomimetic apatites, namely, nanocrystal morphology (plate vs needle-like crystals) and carbonate content, on the bone regeneration potential of CDHA scaffolds in an in vivo canine model. Both ectopic bone formation and scaffold degradation were drastically affected by the nanocrystal morphology after intramuscular implantation. Fine-CDHA foams with needle-like nanocrystals, comparable in size to bone mineral, showed a markedly higher osteoinductive potential and a superior degradation than chemically identical coarse-CDHA foams with larger plate-shaped crystals. These findings correlated well with the superior bone-healing capacity showed by the fine-CDHA scaffolds when implanted intraosseously. Moreover, carbonate doping of CDHA, which resulted in small plate-shaped nanocrystals, accelerated both the intrinsic osteoinduction and the bone healing capacity, and significantly increased the cell-mediated resorption. These results suggest that tuning the chemical composition and the nanostructural features may allow the material to enter the physiological bone remodeling cycle, promoting a tight synchronization between scaffold degradation and bone formation.

Osteogenesis by foamed and 3D-printed nanostructured calcium phosphate scaffolds: Effect of pore architecture.

There is an urgent need of synthetic bone grafts with enhanced osteogenic capacity. This can be achieved by combining biomaterials with exogenous growth factors, which however can have numerous undesired side effects, but also by tuning the intrinsic biomaterial properties. In a previous study, we showed the synergistic effect of nanostructure and pore architecture of biomimetic calcium deficient hydroxyapatite (CDHA) scaffolds in enhancing osteoinduction, i.e. fostering the differentiation of mesenchymal stem cells to bone forming cells. This was demonstrated by assessing bone formation after implanting the scaffolds intramuscularly. The present study goes one step forward, since it analyzes the effect of the geometrical features of the same CDHA scaffolds, obtained either by 3D-printing or by foaming, on the osteogenic potential and resorption behaviour in a bony environment. After 6 and 12 weeks of intraosseous implantation, both bone formation and material degradation had been drastically affected by the macropore architecture of the scaffolds. Whereas nanostructured CDHA was shown to be highly osteoconductive both in the robocast and foamed scaffolds, a superior osteogenic capacity was observed in the foamed scaffolds, which was associated with their higher intrinsic osteoinductive potential. Moreover, they showed a significantly higher cell-mediated degradation than the robocast constructs, with a simultaneous and progressive replacement of the scaffold by new bone. In conclusion, these results demonstrate that the control of macropore architecture is a crucial parameter in the design of synthetic bone grafts, which allows fostering both material degradation and new bone formation. Statement of Significance 3D-printing technologies open new perspectives for the design of patient-specific bone grafts, since they allow customizing the external shape together with the internal architecture of implants. In this respect, it is important to design the appropriate pore geometry to maximize the bone healing capacity of these implants. The present study analyses the effect of pore architecture of nanostructured hydroxyapatite scaffolds, obtained either by 3D-printing or foaming, on the osteogenic potential and scaffold resorption in an in vivo model. While nanostructured hydroxyapatite showed excellent osteoconductive properties irrespective of pore geometry, we demonstrated that the spherical, concave macropores of foamed scaffolds significantly promoted both material resorption and bone regeneration compared to the 3D-printed scaffolds with orthogonal-patterned struts and therefore prismatic, convex macropores.

Osteoinduction by Foamed and 3D-Printed Calcium Phosphate Scaffolds: Effect of Nanostructure and Pore Architecture.

Some biomaterials are osteoinductive, that is, they are able to trigger the osteogenic process by inducing the differentiation of mesenchymal stem cells to the osteogenic lineage. Although the underlying mechanism is still unclear, microporosity and specific surface area (SSA) have been identified as critical factors in material-associated osteoinduction. However, only sintered ceramics, which have a limited range of porosities and SSA, have been analyzed so far. In this work, we were able to extend these ranges to the nanoscale, through the foaming and 3D-printing of biomimetic calcium phosphates, thereby obtaining scaffolds with controlled micro- and nanoporosity and with tailored macropore architectures. Calcium-deficient hydroxyapatite (CDHA) scaffolds were evaluated after 6 and 12 weeks in an ectopic-implantation canine model and compared with two sintered ceramics, biphasic calcium phosphate and ß-tricalcium phosphate. Only foams with spherical, concave macropores and not 3D-printed scaffolds with convex, prismatic macropores induced significant ectopic bone formation. Among them, biomimetic nanostructured CDHA produced the highest incidence of ectopic bone and accelerated bone formation when compared with conventional microstructured sintered calcium phosphates with the same macropore architecture. Moreover, they exhibited different bone formation patterns; in CDHA foams, the new ectopic bone progressively replaced the scaffold, whereas in sintered biphasic calcium phosphate scaffolds, bone was deposited on the surface of the material, progressively filling the pore space. In conclusion, this study demonstrates that the high reactivity of nanostructured biomimetic CDHA combined with a spherical, concave macroporosity allows the pushing of the osteoinduction potential beyond the limits of microstructured calcium phosphate ceramics.

Claudin-16 Deficiency Impairs Tight Junction Function in Ameloblasts, Leading to Abnormal Enamel Formation.

Claudin-16 protein (CLDN16) is a component of tight junctions (TJ) with a restrictive distribution so far demonstrated mainly in the kidney. Here, we demonstrate the expression of CLDN16 also in the tooth germ and show that claudin-16 gene (CLDN16) mutations result in amelogenesis imperfecta (AI) in the 5 studied patients with familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC). To investigate the role of CLDN16 in tooth formation, we studied a murine model of FHHNC and showed that CLDN16 deficiency led to altered secretory ameloblast TJ structure, lowering of extracellular pH in the forming enamel matrix, and abnormal enamel matrix protein processing, resulting in an enamel phenotype closely resembling human AI. This study unravels an association of FHHNC owing to CLDN16 mutations with AI, which is directly related to the loss of function of CLDN16 during amelogenesis. Overall, this study indicates for the first time the importance of a TJ protein in tooth formation and underlines the need to establish a specific dental follow-up for these patients.

In Vivo Osteogenic Potential of Biomimetic Hydroxyapatite/Collagen Microspheres: Comparison with Injectable Cement Pastes.

The osteogenic capacity of biomimetic calcium deficient hydroxyapatite microspheres with and without collagen obtained by emulsification of a calcium phosphate cement paste has been evaluated in an in vivo model, and compared with an injectable calcium phosphate cement with the same composition. The materials were implanted into a 5 mm defect in the femur condyle of rabbits, and bone formation was assessed after 1 and 3 months. The histological analysis revealed that the cements presented cellular activity only in the margins of the material, whereas each one of the individual microspheres was covered with osteogenic cells. Consequently, bone ingrowth was enhanced by the microspheres, with a tenfold increase compared to the cement, which was associated to the higher accessibility for the cells provided by the macroporous network between the microspheres, and the larger surface area available for osteoconduction. No significant differences were found in terms of bone formation associated with the presence of collagen in the materials, although a more extensive erosion of the collagen-containing microspheres was observed.

Augmentation of intramembranous bone in rabbit calvaria using an occlusive barrier in combination with demineralized bone matrix (DBM): a pilot study.

The aim of this study was to histologically evaluate the performance of demineralized bone matrix (DBM) when compared with a blood clot in addition to an occlusive barrier in the bone regeneration process for bone defects in a rabbit model. Prefabricated metallic capsules with 4.5 mm and 3.5 mm dimensions were placed in five adult rabbit skulls. At the right side, the capsule was filled with DBM, and the clot was located on the left side. The barriers were supplied with a 0.5 mm horizontal peripheral flap and a vertical edge, fitting tightly into a circular slit prepared by a trephine in the skull. After a healing period of three months, the animals were sacrificed, and the samples were prepared for histological and histomorphometric analyses after capsule removal. Trabecular and medullar bone percentages were calculated from the different areas of the newly formed bone inside the metallic barriers, and non-parametric statistical analysis was used to describe the findings. The results showed a complete filling of newly formed bone inside the capsules of both groups. Less mature bone tissue was observed in the upper third of all samples, and a higher trabecular area was observed in the samples with DBM. The use of barriers resulted in the augmentation of newly formed bone in a three-month period. However, a higher trabecular area was observed in the barriers filled with DBM.