Exchanging and Releasing Information in Synthetic Digital Polymers Using a Strand-Displacement Strategy.

Toehold-mediated strand displacement (TMSD) was tested as a tool to edit information in synthetic digital polymers. Uniform DNA-polymer biohybrid macromolecules were first synthesized by automated phosphoramidite chemistry and characterized by HPLC, mass spectrometry, and polyacrylamide gel electrophoresis (PAGE). These precursors were diblock structures containing a synthetic poly(phosphodiester) (PPDE) segment covalently attached to a single-stranded DNA sequence. Three types of biohybrids were prepared herein: a substrate containing an accessible toehold as well as input and output macromolecules. The substrate and the input macromolecules contained noncoded PPDE homopolymers, whereas the output macromolecule contained a digitally encoded segment. After hybridization of the substrate with the output, incubation in the presence of the input led to efficient TMSD and the release of the digital segment. TMSD can therefore be used to erase or rewrite information in self-assembled biohybrid superstructures. Furthermore, it was found in this work that the conjugation of DNA single strands to synthetic segments of chosen lengths greatly facilitates the characterization and PAGE visualization of the TMSD process.

Using ion mobility spectrometry to understand signal dilution during tandem mass spectrometry sequencing of digital polymers: Experimental evidence of intramolecular cyclization.

RATIONALE: Optimizing the structure of digital polymers is an efficient strategy to ensure their tandem mass spectrometry (MS/MS) readability. In block-truncated poly(phosphodiester)s, homolysis of C-ON bonds in long chains permits the release of smaller blocks amenable to sequencing. Yet the dissociation behavior of diradical blocks was observed to strongly depend on their charge state. METHODS: Polymers were ionized in negative mode electrospray and activated in-source so that blocks released as primary fragments can be investigated using ion mobility spectrometry (IMS) or sequenced in the post-IMS collision cell. Collision cross sections (CCS) were derived from arrival times using a calibration procedure developed for polyanions using the IMSCal software. A multistep protocol based on quantum methods and classical molecular dynamics was implemented for molecular modeling and calculation of theoretical CCS. RESULTS: Unlike their triply charged homologues, dissociation of diradical blocks at the 2- charge state produces additional fragments, with +1 m/z shift for those holding the nitroxide α-termination and -1 m/z for those containing the carbon-centered radical ω-end. These results suggest cyclization of these diradical species, followed by H• transfer on activated reopening of this cycle. This assumption was validated using IMS resolution of the cyclic/linear isomers and supported by molecular modeling. CONCLUSIONS: Combining IMS with molecular modeling provided new insights into how the charge state of digital blocks influences their dissociation. These results permit to define new guidelines to improve either ionization conditions or the structural design of these digital polymers for best MS/MS readability.

Stability of maxillary expansion osteotomy using patient-specific fixation implants without necessitating removable appliances: a retrospective analysis.

OBJECTIVE: This study aimed to evaluate the long-term stability of surgical maxillary expansion using patient-specific fixation implants (PSFIs) without intraoral retention. MATERIALS AND METHODS: Fifteen patients who had undergone segmented Le Fort I osteotomy and PSFIs with available preoperative (t0) early (t1) and 1-year follow-up computed tomography (CT) scans (t2) were evaluated. The early and 1-year 3D models were superimposed to transfer the bony landmarks; the distances between each pair of landmarks at the different time points were then measured. The distances between the canines and second molars were also measured directly on the CT scans. RESULTS: The achieved maxillary expansions ranged from a median of 4.39 (2.00-6.27) mm at the greater palatine foramina to a median of 2.14 (1.56-2 > 83) mm at the canine level of the palatal bone. One year postoperatively, the changes in skeletal diameters ranged from a median of - 0.53 (- 1.65 to 0.41) mm at the greater palatine foramina (p = 0.12) to 0.17 (- 0.09 to 0.32) mm at the canine level of the palatal bone (p = 0.56). Changes in dental arch diameters ranged from a median of - 0.6 (- 2 to 0.00) mm between the second molars to - 1.3 (- 1.8 to - 0.25) mm between the canines (P < 0.05). CONCLUSION: This study showed the stability of maxillary expansion osteotomy using PSFIs, even without postoperative intraoral retention. CLINICAL RELEVANCE: PSFIs are a reliable method for the surgical treatment of transverse maxillary discrepancy. PFSIs are easy-to-use and improve surgical accuracy.

Macromolecular Information Transfer.

Macromolecular information transfer can be defined as the process by which a coded monomer sequence is communicated from one macromolecule to another. In such a transfer process, the information sequence can be kept identical, transformed into a complementary sequence or even translated into a different molecular language. Such mechanisms are crucial in biology and take place in DNA→DNA replication, DNA→RNA transcription and RNA→protein translation. In fact, there would be no life on Earth without macromolecular information transfer. Mimicking such processes with synthetic macromolecules would also be of major scientific relevance because it would open up new avenues for technological applications (e.g. data storage and processing) but also for the creation of artificial life. In this important context, this minireview summarizes recent research about information transfer in synthetic oligomers and polymers. Medium- and long-term perspectives are also discussed.

Conception and Evaluation of a Library of Cleavable Mass Tags for Digital Polymers Sequencing.

A library of phosphoramidite monomers containing a main-chain cleavable alkoxyamine and a side-chain substituent of variable molar mass (i.e. mass tag) was prepared in this work. These monomers can be used in automated solid-phase phosphoramidite chemistry and therefore incorporated periodically as spacers inside digitally-encoded poly(phosphodiester) chains. Consequently, the formed polymers contain tagged cleavable sites that guide their fragmentation in mass spectrometry sequencing and enhance their digital readability. The spacers were all prepared via a seven steps synthetic procedure. They were afterwards tested for the synthesis and sequencing of model digital polymers. Uniform digitally-encoded polymers were obtained as major species in all cases, even though some minor defects were sometimes detected. Furthermore, the polymers were decoded in pseudo-MS3 conditions, thus confirming the reliability and versatility of the spacers library.

Design of Abiological Digital Poly(phosphodiester)s.

In biological systems, the storage and transfer of genetic information rely on sequence-controlled nucleic acids such as DNA and RNA. It has been realized for quite some time that this property is not only crucial for life but could also be very useful in human applications. For instance, DNA has been actively investigated as a digital storage medium over the past decade. Indeed, the "hard-disk of life" is an obvious choice and a highly optimized material for storing data. Through decades of nucleic acids research, technological tools for parallel synthesis and sequencing of DNA have been readily available. Consequently, it has already been demonstrated that different types of documents (e.g., texts, images, videos, and industrial data) can be stored in chemically synthesized DNA libraries. However, DNA is subject to biological constraints, and its molecular structure cannot be easily varied to match technological needs. In fact, DNA is not the only macromolecule that enables data storage. In recent years, it has been demonstrated that a wide variety of synthetic polymers can also be used for such a purpose. Indeed, modern polymer synthesis allows the preparation of synthetic macromolecules with precisely controlled monomer sequences. Altogether, about a dozens of synthetic digital polymers have already been described, and many more can be foreseen. Among them, sequence-defined poly(phosphodiester)s are one of the most promising options. These polymers are prepared by stepwise phosphoramidite chemistry like chemically synthesized oligonucleotides. However, they are constructed with non-natural building blocks and therefore share almost no structural characteristics with nucleic acids, except phosphate repeat units. Still, they contain readable digital messages that can be deciphered by nanopore sequencing or mass spectrometry sequencing. In this Account, we describe our recent research efforts in synthesizing and sequencing optimal abiological digital poly(phosphodiester)s. A major advantage of these polymers over DNA is that their molecular structure can easily be varied to tune their properties. During the last 5 years, we have engineered the molecular structure of these polymers to adjust crucial parameters such as the storage density, storage capacity, erasability, and readability. Consequently, high-capacity PPDE chains, containing hundreds of bits per chains, can now be synthesized and efficiently sequenced using a routine mass spectrometer. Furthermore, sequencing data can be automatically decrypted with the help of decoding software. This new type of coded matter can also be edited using practical physical triggers such as light and organized in space by programmed self-assembly. All of these recent improvements are summarized and discussed herein.

Covalent Attachment and Detachment by Reactive DESI of Sequence-Coded Polymer Taggants.

The use of sequence-defined polymers is an interesting emerging solution for materials identification and traceability. Indeed, a very large amount of identification sequences can be created using a limited alphabet of coded monomers. However, in all reported studies, sequence-defined taggants are usually included in a host material by noncovalent adsorption or entrapment, which may lead to leakage, aggregation, or degradation. To avoid these problems, sequence-defined polymers are covalently attached in the present work to the mesh of model materials, namely acrylamide hydrogels. To do so, sequence-coded polyurethanes containing a disulfide linker and a terminal methacrylamide moiety are synthesized by stepwise solid-phase synthesis. These methacrylamide macromonomers are afterward copolymerized with acrylamide and bisacrylamide in order to achieve cross-linked hydrogels containing covalently-bound polyurethane taggants. It is shown herein that these taggants can be selectively detached from the hydrogel mesh by reactive desorption electrospray ionization. Using dithiothreitol the disulfide linker that links the taggant to the gel can be selectively cleaved. Ultimately, the released taggants can be decoded by tandem mass spectrometry.

Receptor for Advanced Glycation End-Products Promotes Activation of Alveolar Macrophages through the NLRP3 Inflammasome/TXNIP Axis in Acute Lung Injury.

The roles of thioredoxin-interacting protein (TXNIP) and receptor for advanced glycation end-products (RAGE)-dependent mechanisms of NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome-driven macrophage activation during acute lung injury are underinvestigated. Cultured THP-1 macrophages were treated with a RAGE agonist (S100A12), with or without a RAGE antagonist; cytokine release and intracytoplasmic production of reactive oxygen species (ROS) were assessed in response to small interfering RNA knockdowns of TXNIP and NLRP3. Lung expressions of TXNIP and NLRP3 and alveolar levels of IL-1ß and S100A12 were measured in mice after acid-induced lung injury, with or without administration of RAGE inhibitors. Alveolar macrophages from patients with acute respiratory distress syndrome and from mechanically ventilated controls were analyzed using fluorescence-activated cell sorting. In vitro, RAGE promoted cytokine release and ROS production in macrophages and upregulated NLRP3 and TXNIP mRNA expression in response to S100A12. TXNIP inhibition downregulated NLRP3 gene expression and RAGE-mediated release of IL-1ß by macrophages in vitro. In vivo, RAGE, NLRP3 and TXNIP lung expressions were upregulated during experimental acute lung injury, a phenomenon being reversed by RAGE inhibition. The numbers of cells expressing RAGE, NLRP3 and TXNIP among a specific subpopulation of CD16+CD14+CD206- ("pro-inflammatory") alveolar macrophages were higher in patients with lung injury. This study provides a novel proof-of-concept of complex RAGE-TXNIP-NLRP3 interactions during macrophage activation in acute lung injury.

Chain Entropy Beats Hydrogen Bonds to Unfold and Thread Dialcohol Phosphates inside Cyanostar Macrocycles To Form [3]Pseudorotaxanes.

The recognition of substituted phosphates underpins many processes including DNA binding, enantioselective catalysis, and recently template-directed rotaxane synthesis. Beyond ATP and a few commercial substrates, however, little is known about how substituents effect organophosphate recognition. Here, we examined alcohol substituents and their impact on recognition by cyanostar macrocycles. The organophosphates were disubstituted by alcohols of various chain lengths, dipropanol, dihexanol, and didecanol phosphate, each accessed using modular solid-phases syntheses. Based on the known size-selective binding of phosphates by π-stacked dimers of cyanostars, threaded [3]pseudorotaxanes were anticipated. While seen with butyl substituents, pseudorotaxane formation was disrupted by competitive OH···O- hydrogen bonding between both terminal hydroxyls and the anionic phosphate unit. Crystallography also showed formation of a backfolded propanol conformation resulting in an 8-membered ring and a perched cyanostar assembly. Motivated by established entropic penalties accompanying ring formation, we reinstated [3]pseudorotaxanes by extending the size of the substituent to hexanol and decanol. Chain entropy overcomes the enthalpically favored OH···O- contacts to favor random-coil conformations required for seamless, high-fidelity threading of dihexanol and didecanol phosphates inside cyanostars. These studies highlight how chain length and functional groups on phosphate's substituents can be powerful design tools to regulate binding and control assembly formation during phosphate recognition.

Precise Alkoxyamine Design to Enable Automated Tandem Mass Spectrometry Sequencing of Digital Poly(phosphodiester)s.

A major step towards reliable reading of information coded in the sequence of long poly(phosphodiester)s was previously achieved by introducing an alkoxyamine spacer between information sub-segments. However, MS/MS decoding had to be performed manually to safely identify useful fragments of low abundance compared to side-products from the amide-based alkoxyamine used. Here, alternative alkoxyamines were designed to prevent side-reactions and enable automated MS/MS sequencing. Different styryl-TEMPO spacers were prepared to increase radical delocalization and stiffness of the structure. Their dissociation behavior was investigated by EPR and best results were obtained with spacers containing in-chain benzyl ring, with no side-reaction during synthesis or sequencing. Automated decoding of these polymers was performed using the MS-DECODER software, which interprets fragmentation data recorded for each sub-segment and re-align them in their original order based on location tags.

Optimal conditions for tandem mass spectrometric sequencing of information-containing nitrogen-substituted polyurethanes.

RATIONALE: To prevent solubility issues faced with sequence-defined polyurethanes, a new family of digital polyurethanes was conceived with the alkyl coding chain held by the carbamate nitrogen (N) atom and CH3 instead of OH as the ϖ termination. This led to different dissociation mechanisms that were explored prior to optimizing tandem mass spectrometric (MS/MS) sequencing. METHODS: N-Substituted polyurethanes (N-R PUs) were dissolved in methanol and subjected to collision-induced dissociation (CID) as deprotonated chains in the negative ion mode, and as ammonium and sodium adducts in the positive ion mode, using electrospray ionization (ESI) as the ionization technique. Their dissociation behavior was thoroughly investigated using a quadrupole time-of-flight (QTOF) instrument in order to provide accurate mass measurements to support proposed fragmentation mechanisms. RESULTS: While O-(CO) bonds were broken in N-H PUs, the CH2 -O linkage between repeating units was cleaved upon CID of N-R PUs. This main process occurred either from deprotonated molecules or from cationized chains but was followed by different rearrangements, producing up to four product ion series. Yet, MS/MS spectra could be drastically simplified for precursor ions having their acidic α group methylated, as was found to spontaneously occur upon storage in methanol. CONCLUSIONS: Using experimental conditions aimed at avoiding any reactive proton in precursor ions (no acidic end-groups and alkali adduction), full coverage sequence of N-R PUs was successfully achieved with the single ion series observed in MS/MS, opening a promising perspective for reading large amounts of information stored in these dyad-encoded polymers.

Selective Bond Cleavage in Informational Poly(Alkoxyamine Phosphodiester)s.

The collision-induced dissociation (CID) of sequence-defined poly(alkoxyamine phosphodiester)s is studied by electrospray ionization mass spectrometry. These informational polymers are synthesized using three different nitroxide building blocks, namely proxyl-, SG1-, and TEMPO-derivatives. For a polymer containing TEMPO- and SG1-based main chain alkoxyamines, it is found that both types of alkoxyamines break in CID tandem mass spectrometry (MS/MS). However, SG1-sites are preferentially cleaved and this predominance can be increased by reducing collision energy, even though selective bond fragmentation is not observed. On the other hand, for a polymer containing proxyl- and SG1-alkoxyamines, selective bond cleavage is observed at all studied collision energies. The SG1-alkoxyamines can be first cleaved in MS/MS conditions and secondly the proxyl-alkoxyamines in pseudo-MS3 conditions. These results open up interesting new avenues for the design of readable, erasable or programmable informational polymers.

Prediction of Post-Traumatic Enophthalmos Based on Orbital Volume Measurements: A Systematic Review.

PURPOSE: Enophthalmos greater than 2 mm should be considered clinically relevant and can be responsible for esthetic and functional morbidity. The difficulty has always been the best method to use to accurately determine when the orbital wall displacement will lead to clinically relevant enophthalmos. None of the currently used techniques is able to accurately predict for post-traumatic enophthalmos (PE). The aim of the present study was to systematically review the use of orbital volumetric tools in the prediction of PE after orbital fracture. MATERIALS AND METHODS: The terms searched in each database were "(orbital volumetry) and enophthalmos," "volumetry and enophthalmos," "volume and enophthalmos," and "volumetric and enophthalmos." The relationship between PE and the orbital volume (OV) was assessed. RESULTS: The initial search yielded 346 results. Of the 346 studies, 14 were included and analyzed. Every study reported a different numerical relationship between the OV and PE, with a mean enophthalmos of 0.80 mm after a 1-cm3 increase in the OV. CONCLUSIONS: The present review found that most studies concluded that a direct relationship exists between the OV and PE and defined the degree of PE in relation to the OV expansion. Enophthalmos assessment using radiologic evaluation provides increased accuracy and reproducibility compared with clinical measurement using an exophthalmometer. It has been notoriously difficult to determine when orbital wall displacement will lead to clinically relevant enophthalmos. Measurement of the OV could have a role in the decision for surgical or conservative treatment.

Standard and Customized Alloplastic Facial Implants Refining Orthognathic Surgery: Outcome Evaluation.

PURPOSE: Conventional orthognathic osteotomies provide appropriate functional outcomes but might be unable to correct midface deficiency, achieve a satisfactory outcome in asymmetrical cases, or allow sufficient chin advancement. We evaluated the outcome of both standard and customized facial high-density porous polyethylene implants used to refine the cosmetic outcome of orthognathic surgery. PATIENTS AND METHODS: We implemented a retrospective study. The sample was composed of all patients who underwent facial alloplastic augmentation between June 2011 and October 2018 in our department. The complication rate was recorded after a mean follow-up period of 41 months postoperatively, and patient satisfaction was assessed through a qualitative evaluation based on an 11-item questionnaire. RESULTS: The sample was composed of 24 implants placed in 14 patients: 13 mandibular angle implants, among which 4 were customized; 8 malar implants; and 3 chin implants. No physical complications such as hematoma, infection, migration, or hypoesthesia were observed. Two implants had to be removed because of early unsatisfactory esthetic outcomes. Of 14 patients, 11 answered our questionnaire. Eighty-two percent strongly agreed that the overall outcome was satisfactory. CONCLUSIONS: The results of this study confirm the low physical complication rate described in the literature, and the esthetic complication rate remains lower than the rates observed in previous reports. A high satisfaction rate was found among patients. The lowest mean satisfaction score was noted for appropriate implant symmetry (3.5), whereas the highest mean satisfaction score (3.8) was achieved when using customized implants. If standard high-density porous polyethylene implants appear to be relevant adjuncts to orthognathic surgery, customized implants seem to achieve higher satisfaction, although their prohibitive cost should be considered.

Damage and Repair in Informational Poly(N-substituted urethane)s.

The degradation and repair of uniform sequence-defined poly(N-substituted urethane)s was studied. Polymers containing an ω-OH end-group and only ethyl carbamate main-chain repeat units rapidly degrade in NaOH solution through an ω→α depolymerization mechanism with no apparent sign of random chain cleavage. The degradation mechanism is not notably affected by the nature of the side-chain N-substituents and took place for all studied sequences. On the other hand, depolymerization is significantly influenced by the molecular structure of the main-chain repeat units. For instance, hexyl carbamate main-chain motifs block unzipping and can therefore be used to control the degradation of specific sequence sections. Interestingly, the partially degraded polymers can also be repaired; for example by using a combination of N,N'-disuccinimidyl carbonate with a secondary amine building-block. Overall, these findings open up interesting new avenues for chain-healing and sequence editing.

Revealing Data Encrypted in Sequence-Controlled Poly(Alkoxyamine Phosphodiester)s by Combining Ion Mobility with Tandem Mass Spectrometry.

The defined sequence of two comonomers in sequence-controlled macromolecules can be used to store binary information which is further decoded by MS/MS sequencing. In order to achieve the full sequence coverage requested for reliable decoding, the structure of these polymers can be optimized to minimize their dissociation extent, as shown for poly(alkoxyamine phosphodiester)s (PAPs) where weak alkoxyamine bonds were introduced in each repeating unit to make all phosphate groups MS/MS silent. However, for secret communications, a too high MS/MS readability could be a drawback. In this context, the design of PAPs was further optimized in this work to also include a decrypting key based on slight variation of a fragment collision cross section. This was achieved by employing two different nitroxides to build the alkoxyamine moiety, each containing a coding alkyl segment of the same mass but different architectures. As a result, the digital sequence determined from primary fragments observed in MS/MS had to be decrypted according to appropriate rules that depend on the drift times measured by ion mobility spectrometry for repeating units released as secondary product ions.

A New Software Suite in Orthognathic Surgery : Patient Specific Modeling, Simulation and Navigation.

Orthognathic surgery belongs to the scope of maxillofacial surgery. It treats dentofacial deformities consisting in discrepancy between the facial bones (upper and lower jaws). Such impairment affects chewing, talking, and breathing and can ultimately result in the loss of teeth. Orthognathic surgery restores facial harmony and dental occlusion through bone cutting, repositioning, and fixation. However, in routine practice, we face the limitations of conventional tools and the lack of intraoperative assistance. These limitations occur at every step of the surgical workflow: preoperative planning, simulation, and intraoperative navigation. The aim of this research was to provide novel tools to improve simulation and navigation. We first developed a semiautomated segmentation pipeline allowing accurate and time-efficient patient-specific 3D modeling from computed tomography scans mandatory to achieve surgical planning. This step allowed an improvement of processing time by a factor of 6 compared with interactive segmentation, with a 1.5-mm distance error. Next, we developed a software to simulate the postoperative outcome on facial soft tissues. Volume meshes were processed from segmented DICOM images, and the Bullet open source mechanical engine was used together with a mass-spring model to reach a postoperative simulation accuracy <1 mm. Our toolset was completed by the development of a real-time navigation system using minimally invasive electromagnetic sensors. This navigation system featured a novel user-friendly interface based on augmented virtuality that improved surgical accuracy and operative time especially for trainee surgeons, therefore demonstrating its educational benefits. The resulting software suite could enhance operative accuracy and surgeon education for improved patient care.

Retrieving a displaced third molar from the infratemporal fossa: case report of a minimally invasive procedure.

BACKGROUND: The appropriate management of postoperative complication of wisdom teeth removal is of utmost importance as it can result in legal procedures. The accidental displacement of a maxillary third molar in the infratemporal fossa (ITF), is a rare complication that can occur even with experienced surgeons. The numerous retrieval techniques reported are invasive and provide an unpredictable access. Our aim was to achieve the safe and swift retrieval of the tooth displaced to an area of such complex anatomy. CASE PRESENTATION: We describe the case of a 17-year-old female patient whose right upper third molar was accidentally pushed upward to the ITF and became unreachable. Retrieval based on interventional radiology using the CT-guided placement of a bone trocar above the displaced tooth was successfully performed. The postoperative course was uneventful. CONCLUSIONS: CT scan assisted interventional radiology provides both, real-time assessment of the tooth position through image refreshment, and steady stabilization of the displaced tooth. Therefore, it allows a safe and non-traumatic retrieval with a time-efficient procedure achieved through a minimally-invasive approach with inconspicuous scaring. We believe that such a procedure is an interesting treatment option for optimal outpatient care. To our knowledge, no such case has been previously described.

Synthesis of Macromolecules Containing Phenylalanine and Aliphatic Building Blocks.

Aiming at developing efficient interfacial agents for fiber-reinforced composite materials, macromolecules are designed to have different components able to stick to the fiber and be compatible with the polymer matrix, respectively. Herein, macromolecules are prepared by solid-phase synthesis considering phenylalanine residues to promote adsorption of the macromolecule on aramid fibers and aliphatic building blocks to interact with a hydrophobic polymer matrix. Using phenylalanine as building block for the preparation of macromolecules by iterative synthesis has been shown to be challenging. Thus, the screening of various parameters for the optimization of the synthesis of these macromolecules is discussed in this communication. A preliminary thermal study by thermal gravimetric analysis is conducted to evaluate their thermal stability.

Temporomandibular Joint Regenerative Medicine.

The temporomandibular joint (TMJ) is an articulation formed between the temporal bone and the mandibular condyle which is commonly affected. These affections are often so painful during fundamental oral activities that patients have lower quality of life. Limitations of therapeutics for severe TMJ diseases have led to increased interest in regenerative strategies combining stem cells, implantable scaffolds and well-targeting bioactive molecules. To succeed in functional and structural regeneration of TMJ is very challenging. Innovative strategies and biomaterials are absolutely crucial because TMJ can be considered as one of the most difficult tissues to regenerate due to its limited healing capacity, its unique histological and structural properties and the necessity for long-term prevention of its ossified or fibrous adhesions. The ideal approach for TMJ regeneration is a unique scaffold functionalized with an osteochondral molecular gradient containing a single stem cell population able to undergo osteogenic and chondrogenic differentiation such as BMSCs, ADSCs or DPSCs. The key for this complex regeneration is the functionalization with active molecules such as IGF-1, TGF-ß1 or bFGF. This regeneration can be optimized by nano/micro-assisted functionalization and by spatiotemporal drug delivery systems orchestrating the 3D formation of TMJ tissues.