Translating Dental, Oral, and Craniofacial Regenerative Medicine Innovations to the Clinic through Interdisciplinary Commercial Translation Architecture.

Few university-based regenerative medicine innovations in the dental, oral, and craniofacial (DOC) space have been commercialized and affected clinical practice in the United States. An analysis of the commercial translation literature and National Institute for Dental and Craniofacial Research's (NIDCR's) portfolio identified barriers to commercial translation of university-based DOC innovations. To overcome these barriers, the NIDCR established the Dental Oral Craniofacial Tissue Regeneration Consortium. We provide generalized strategies to inform readers how to bridge the "valley of death" and more effectively translate DOC technologies from the research laboratory or early stage company environment to clinical trials and bring needed innovations to the clinic. Three valleys of death are covered: 1) from basic science to translational development, 2) from translational technology validation to new company formation (or licensing to an existing company), and 3) from new company formation to scaling toward commercialization. An adapted phase-gate model is presented to inform DOC regenerative medicine teams how to involve regulatory, manufacturability, intellectual property, competitive assessments, business models, and commercially oriented funding mechanisms earlier in the translational development process. An Industrial Partners Program describes how to conduct market assessments, industry maps, business development processes, and industry relationship management methods to sustain commercial translation through the later-stage valley of death. Paramount to successfully implementing these methods is the coordination and collaboration of interdisciplinary teams around specific commercial translation goals and objectives. We also provide several case studies for translational projects with an emphasis on how they addressed DOC biomaterials for tissue regeneration within a rigorous commercial translation development environment. These generalized strategies and methods support innovations within a university-based and early stage company-based translational development process, traversing the many funding gaps in dental, oral, and craniofacial regenerative medicine innovations. Although the focus is on shepherding technologies through the US Food and Drug Administration, the approaches are applicable worldwide.

The role of inflammation modulation in dental pulp regeneration.

A vital and healthy dental pulp (DP) is required for teeth to remain functional throughout a lifespan . Appreciating its value for the tooth, the regeneration of the DP is a highly researched goal. While inflammation of the DP marks the beginning of an eventual necrosis, it is also the prerequisite for the regenerative events of neovascularisation, stem cells mobilisation and reparative dentine deposition. In the light of a pro-regenerative inflammatory process, the present review discusses the role of macrophage population shift from pro- to anti-inflammatory in reversible versus irreversible pulpitis, while also analysing the overlooked contribution of pulp innervation and locally derived neuropeptides to the process. Then, the currently practiced (pulp capping and revascularisation) and researched (cells transplantation and cell homing) approaches for DP regeneration are discussed. Focusing on the role of cell homing in modulating inflammation, some potential strategies are highlighted to harness the inflammatory process for DP regeneration, mainly by reversing inflammation through macrophage induction. Next, some potential clinical applications are discussed - especially with capping materials - that could boost macrophage polarisation and complement system activation. Finally, current challenges facing the regeneration of the DP are presented, while underlining the importance of promoting an anti-inflammatory environment conducive to a regenerative process.

[Advance directives in hospital practice: A matter of concern for every healthcare professional and consumer]. / Les directives anticipées à l'hôpital : l'affaire de tous ?

INTRODUCTION: In France, Leonetti and Claeys-Leonetti laws relating to patients' rights and end-of-life practice have introduced the advance healthcare directives (ADs). Although family doctor's role is important in initiating discussions regarding AD, hospital healthcare professionals should also be concerned by the health care planning laws. METHODS: A descriptive, quantitative and qualitative study was conducted in Paris Saint-Joseph hospital to evaluate the knowledge of nursing personnel regarding ADs. Among healthcare professionals present on 02/06/2016 and agreeing to participate, 50 non-medical caregivers and 50 doctors were randomly selected and took part in this survey. Three trainee lawyers conducted interviews, recorded and anonymized them. The Nvivo software analyzed the qualitative part of the results. RESULTS: Only 10% of healthcare professionals knew these legal and ethical issues in health care. Most caregivers were not in favor of informing all patients admitted to a hospital (hospitalized patients or patients received consultations). For 44%, only hospitalized end-of-life patients should be informed about ADs. For 76% of the people questioned, family doctor has a unique position to guide the patient on the preparation and registration of living wills. In hospital stay, the nurse was proposed by 52% of the staff as the preferred caregiver for AD communication, as part of an interdisciplinary healthcare team approach. Finally, the clear majority of caregivers (85%), called for discussions and documentation about ADs, and end-of-life training. CONCLUSION: Advance directives remain poorly known in the hospital, 12 years after the first Leonetti law. The attitude of professionals about ADs is not homogenous but interest for the subject is obvious in the vast majority of caregivers. The results of this survey highlighted that discussions and documentation about ADs as well as training on end-of-life patient care are essential.

Induction of M2 Macrophages Prevents Bone Loss in Murine Periodontitis Models.

Periodontitis is characterized by the progressive destruction of tooth-supporting alveolar bone, which is mainly caused by chronic inflammation in response to persistent bacterial insult. It has recently become clear that the pathogenesis of periodontitis is associated with a high ratio of proinflammatory M1 (classically activated) macrophages to anti-inflammatory M2 (alternatively activated). To decrease the inflammatory activity, we locally delivered the C-C motif chemokine ligand 2 (CCL2) using controlled-release microparticles (MPs). CCL2 is known to promote chemotaxis of M0 or M2 phenotype macrophages to the inflamed site and induce M2 phenotype polarization locally. Our in vitro data showed that CCL2 increased the number of M2 phenotype macrophages, decreased TNF-α secretion, and enhanced chemotaxis of RAW264.7 cells toward CCL2 MPs. Moreover, we induced periodontal disease in 2 animal models through inoculation of Porphyromonas gingivalis and ligature around the murine molar. Micro-computed tomography analysis showed significant reduction of alveolar bone loss in the CCL2 MP treatment group when compared with a blank MP group and a no-treatment periodontitis group in both models. Immunohistologic analysis showed a significant increase in the M2 phenotype subset and a decrease in the M1 phenotype subset in the CCL2 MP group of the P. gingivalis-induced model. Also, in both models, tartrate-resistant acidic phosphatase staining showed significantly fewer numbers of osteoclasts in the CCL2 MP group in alveolar bone area. Moreover, quantitative polymerase chain reaction results showed a significant increase in IL-1RA (interleukin 1 receptor antagonist) mRNA expression and a decrease in RANKL (receptor activator of nuclear factor kappa-Β ligand) mRNA expression in the CCL2 MP group in the ligature model. In summary, manipulation of endogenous M2 phenotype macrophages with CCL2 MPs decreased the M1 phenotype:M2 phenotype ratio and prevented alveolar bone loss in mouse periodontitis models. The delivery of CCL2 MPs provides a novel approach to treat periodontal disease.

Decellularized Swine Dental Pulp Tissue for Regenerative Root Canal Therapy.

In the current theme of dental pulp regeneration, biological and synthetic scaffolds are becoming a potential therapy for pulp revitalization. The goal is to provide a suitable environment for cellular infiltration, proliferation, and differentiation. The extracellular matrix (ECM) represents a natural scaffold material resembling the native tissue chemical and mechanical properties. In the past few years, ECM-based scaffolds have shown promising results in terms of progenitor cells recruitment, promotion of constructive remodeling, and modulation of host response. These properties make ECM-derived scaffolds an ideal candidate for pulp regenerative therapy. Development of strategies for clinically relevant tissue engineering using dental pulp extracellular matrix (DP-ECM) can provide an alternative to conventional root canal treatment. In this work, we successfully decellularized ECM derived from porcine dental pulp. The resulting scaffold was characterized using immunostaining (collagen type I, dentin matrix protein 1, dentin sialoprotein, and Von Willebrand factor) and enzyme-linked immunosorbent assay (transforming growth factor ß, vascular endothelial growth factor, and basic fibroblast growth factor) for extracellular proteins where the ECM retained its proteins and significant amount of growth factors. Furthermore, a pilot in vivo study was conducted where the matrix was implanted for 8 wk in a dog root canal model. Our in vitro and preliminary in vivo data show that the decellularized ECM supports cellular infiltration together with the expression of pulp-dentin and vascular markers (DSP and CD31) compared to the controls. Herein, we show the feasibility to produce a decellularized ECM scaffold and validate the concept of using ECM-based scaffolds for pulp regeneration.

RANKL Triggers Treg-Mediated Immunoregulation in Inflammatory Osteolysis.

The chronic inflammatory immune response triggered by the infection of the tooth root canal system results in the local upregulation of RANKL, resulting in periapical bone loss. While RANKL has a well-characterized role in the control of bone homeostasis/pathology, it can play important roles in the regulation of the immune system, although its possible immunoregulatory role in infectious inflammatory osteolytic conditions remains largely unknown. Here, we used a mouse model of infectious inflammatory periapical lesions subjected to continuous or transitory anti-RANKL inhibition, followed by the analysis of lesion outcome and multiple host response parameters. Anti-RANKL administration resulted in arrest of bone loss but interfered in the natural immunoregulation of the lesions observed in the untreated group. RANKL inhibition resulted in an unremitting proinflammatory response, persistent high proinflammatory and effector CD4 response, decreased regulatory T-cell (Treg) migration, and lower levels of Treg-related cytokines IL-10 and TGFb. Anti-RANKL blockade impaired the immunoregulatory process only in early disease stages, while the late administration of anti-RANKL did not interfere with the stablished immunoregulation. The impaired immunoregulation due to RANKL inhibition is characterized by increased delayed-type hypersensitivity in vivo and T-cell proliferation in vitro to the infecting bacteria, which mimic the effects of Treg inhibition, reinforcing a possible influence of RANKL on Treg-mediated suppressive response. The adoptive transfer of CD4+FOXp3+ Tregs to mice receiving anti-RANKL therapy restored the immunoregulatory capacity, attenuating the inflammatory response in the lesions, reestablishing normal T-cell response in vivo and in vitro, and preventing lesion relapse upon anti-RANKL therapy cessation. Therefore, while RANKL inhibition efficiently limited the periapical bone loss, it promoted an unremitting host inflammatory response by interfering with Treg activity, suggesting that this classic osteoclastogenic mediator plays a role in immunoregulation.

Poly (glycerol sebacate) elastomer supports bone regeneration by its mechanical properties being closer to osteoid tissue rather than to mature bone.

Mechanical load influences bone structure and mass. Arguing the importance of load-transduction, we investigated the mechanisms inducing bone formation using an elastomeric substrate. We characterized Poly (glycerol sebacate) (PGS) in vitro for its mechanical properties, compatibility with osteoprogenitor cells regarding adhesion, proliferation, differentiation under compression versus static cultures and in vivo for the regeneration of a rabbit ulna critical size defect. The load-transducing properties of PGS were compared in vitro to a stiffer poly lactic-co-glycolic-acid (PLA/PGA) scaffold of similar porosity and interconnectivity. Under cyclic compression for 7days, we report focal adhesion kinase overexpression on the less stiff PGS and upregulation of the transcription factor Runx2 and late osteogenic markers osteocalcin and bone sialoprotein (1.7, 4.0 and 10.0 folds increase respectively). Upon implanting PGS in the rabbit ulna defect, histology and micro-computed tomography analysis showed complete gap bridging with new bone by the PGS elastomer by 8weeks while minimal bone formation was seen in empty controls. Immunohistochemical analysis demonstrated the new bone to be primarily regenerated by recruited osteoprogenitors cells expressing periostin protein during early phase of maturation similar to physiological endochondral bone development. This study confirms PGS to be osteoconductive contributing to bone regeneration by recruiting host progenitor/stem cell populations and as a load-transducing substrate, transmits mechanical signals to the populated cells promoting differentiation and matrix maturation toward proper bone remodeling. We hence conclude that the material properties of PGS being closer to osteoid tissue rather than to mineralized bone, allows bone maturation on a substrate mechanically closer to where osteoprogenitor/stem cells differentiate to develop mature load-bearing bone. SIGNIFICANCE OF SIGNIFICANCE: The development of effective therapies for bone and craniofacial regeneration is a foremost clinical priority in the mineralized tissue engineering field. Currently at risk are patients seeking treatment for craniofacial diseases, traumas and disorders including birth defects such as cleft lip and palate, (1 in 525 to 714 live births), craniosynostosis (300-500 per 1,000,000 live births), injuries to the head and face (20 million ER visits per year), and devastating head and neck cancers (8000 deaths and over 30,000 new cases per year). In addition, approximately 6.2 million fractures occur annually in the United States, of which 5-10% fail to heal properly, due to delayed or non-union [1], and nearly half of adults aged 45-65 have moderate to advanced periodontitis with associated alveolar bone loss, which, if not reversed, will lead to the loss of approximately 6.5 teeth/individual [2]. The strategies currently available for bone loss treatment largely suffer from limitations in efficacy or feasibility, necessitating further development and material innovation. Contemporary materials systems themselves are indeed limited in their ability to facilitate mechanical stimuli and provide an appropriate microenvironment for the cells they are designed to support. We propose a strategy which aims to leverage biocompatibility, biodegradability and material elasticity in the creation of a cellular niche. Within this niche, cells are mechanically stimulated to produce their own extracellular matrix. The hypothesis that mechanical stimuli will enhance bone regeneration is supported by a wealth of literature showing the effect of mechanical stimuli on bone cell differentiation and matrix formation. Using mechanical stimuli, to our knowledge, has not been explored in vivo in bone tissue engineering applications. We thus propose to use an elastomeric platform, based on poly(glycerol sebacate (PGS), to mimic the natural biochemical environment of bone while enabling the transmission of mechanical forces. In this study we report the material's load-transducing ability as well as falling mechanically closer to bone marrow and osteoid tissue rather than to mature bone, allowed osteogenesis and bone maturation. Defying the notion of selecting bone regeneration scaffolds based on their relative mechanical comparability to mature bone, we consider our results in part novel for the new application of this elastomer and in another fostering for reassessment of the current selection criteria for bone scaffolds.

Restoring host-microbe homeostasis via selective chemoattraction of Tregs.

The disruption of host-microbe homeostasis at the site of periodontal disease is considered a key factor for disease initiation and progress. While the downstream mechanisms responsible for the tissue damage per se are relatively well-known (involving various patterns of immune response operating toward periodontal tissue destruction), we are only beginning to understand the complexity of host-microbe interactions in the periodontal environment. Unfortunately, most of the research has been focused on the disruption of host-microbe homeostasis instead of focusing on the factors responsible for maintaining homeostasis. In this context, regulatory T-cells (Tregs) comprise a CD4+FOXp3 +T-cell subset with a unique ability to regulate other leukocyte functions to avoid excessive immune activation and its pathological consequences. Tregs act as critical determinants of host-microbe homeostasis, as well as determinants of a balanced host response after the disruption of host-microbe homeostasis by pathogens. In periodontitis, Tregs play a protective role, with their natural recruitment being responsible for conversion of active into inactive lesions. With controlled-release technology, it is now possible to achieve a selective chemoattraction of Tregs to periodontal tissues, attenuating experimental periodontitis evolution due to the local control of inflammatory immune response and the generation of a pro-reparative environment.

Scaffoldless tissue-engineered dental pulp cell constructs for endodontic therapy.

A major cause of apical periodontitis after endodontic treatment is the bacterial infiltration which could have been challenged by the presence of a vital pulp. In this study, self-assembled, scaffoldless, three-dimensional (3D) tissues were engineered from dental pulp cells (DPCs) and assessed as a device for pulp regeneration. These engineered tissues were placed into the canal space of human tooth root segments that were capped on one end with calcium phosphate cement, and the entire system was implanted subcutaneously into mice. Histological staining indicated that after three- and five-month implantations, tooth roots containing 3D scaffoldless engineered tissues maintained a cellular, fibrous tissue throughout, whereas empty tooth roots remained predominantly empty. Immunostaining indicated that the tissue found in the root canals containing scaffoldless DPC engineered tissues was vascular, as characterized by the expression of CD31, and contained odontoblast-like cells organized along the length of the root wall as assessed by immunostaining for dentin sialoprotein. This study shows that 3D self-assembled scaffoldless DPC engineered tissues can regenerate a vital dental pulp-like tissue in a tooth root canal system and are therefore promising for endodontic therapy.

Biomimetic coating of magnesium alloy for enhanced corrosion resistance and calcium phosphate deposition.

Degradable metals have been suggested as biomaterials with revolutionary potential for bone-related therapies. Of these candidate metals, magnesium alloys appear to be particularly attractive candidates because of their non-toxicity and outstanding mechanical properties. Despite their having been widely studied as orthopedic implants for bone replacement/regeneration, their undesirably rapid corrosion rate under physiological conditions has limited their actual clinical application. This study reports the use of a novel biomimetic peptide coating for Mg alloys to improve the alloy corrosion resistance. A 3DSS biomimetic peptide is designed based on the highly acidic, bioactive bone and dentin extracellular matrix protein, phosphophoryn. Surface characterization techniques (scanning electron microscopy, energy dispersive X-ray spectroscopy and diffuse-reflectance infrared spectroscopy) confirmed the feasibility of coating the biomimetic 3DSS peptide onto Mg alloy AZ31B. The 3DSS peptide was also used as a template for calcium phosphate deposition on the surface of the alloy. The 3DSS biomimetic peptide coating presented a protective role of AZ31B in both hydrogen evolution and electrochemical corrosion tests.

Efficient bone formation by gene transfer of human LIM mineralization protein-3.

LIM mineralization protein (LMP) is a novel positive regulator of the osteoblast differentiation program. In humans, three different LMP splice variants have been identified: LMP-1, LMP-2, and LMP-3. Gene transfer of human LMP-1 (hLMP-1) induces expression of genes involved in bone formation, including certain bone morphogenetic proteins (BMPs), promotes bone nodule formation in vitro, ectopic bone formation in vivo, and is therapeutic in animal models of posterior thoracic and lumbar spine fusion. To examine the osteoinductive properties of the LMP-3 in vitro and in vivo, we have generated plasmid and adenoviral vectors expressing codon-optimized hLMP-3. Here we demonstrate that gene transfer of hLMP-3 induces expression of the bone-specific genes osteocalcin, osteopontin, and bone sialoprotein and induced bone mineralization in preosteoblastic and fibroblastic cells. We also demonstrate that hLMP-3 is able to induce bone mineralization and the expression of the bone-specific genes, BMP-2, OSX, RunX2, and alkaline phosphatase in human mesenchymal stem cells in a dose-dependent manner. Finally, we demonstrate that direct gene transfer of hLMP-3 into murine skeletal muscle results in ectopic bone formation more efficiently than BMP-2. These results demonstrate that hLMP-3 gene transfer can be used to promote bone formation in cell culture and in vivo as or more efficiently than BMP-2, thus establishing feasibility and efficacy of direct gene delivery of hLMP-3 to produce bone in vivo. These results suggest that gene transfer of hLMP-3 could be developed as a bone-inductive therapeutic agent for clinical applications.

Critical aspects of tissue-engineered therapy for bone regeneration.

Recent advances in bone tissue engineering are established on the understanding of an engineered scaffold, the molecular milieu within the osteogenic site, and the cell(s) predisposed to an osteogenic lineage. Advances in the incorporation of a generative vehicle into a skeletal defect require temporal and spatial distribution of the scaffold, growth factor, and cell compatible with enhanced bone healing. Monitoring events culminating in osteogenesis has focused on phenotypic and intracellular indicators. Phenotypic and intracellular indicators include the presence of receptors and intracellular signals that enable cell proliferation and differentiation. Progress in the areas of scaffold design, growth factor utilization, bone cell lineage, and intracellular signaling are reviewed.

Therapeutic potential of bone morphogenetic proteins.

Recently, there has been substantial progress in the area of bone morphogenetic protein (BMP) research. This review serves as an up-to-date summary of the history of BMPs, the mechanisms of BMP signalling and the role of BMPs in adipose, kidney, liver, bone and nervous system. The potential of BMPs as therapeutic agents will also be discussed.

Gene therapy approaches for modulating bone regeneration.

Following injury, bone has the ability to regenerate itself to a form and function nearly indistinguishable from the pre-injury state. However, if the injury is beyond a critical limit, recovery will not occur without therapeutic interventions. Autografts and implants with banked bone continue as the treatments of choice, although each exhibits limitations and liabilities. Alternatives have included the utilization of bone-graft substitutes that may incorporate bone derivatives and soluble signaling molecules such as mitogens and morphogens. In addition, an evolving treatment modality, gene therapy, offers an exciting avenue for bone regeneration. This review presents some of the current concepts for developing a rational gene therapy approach in bone regeneration.

Properties of the (DSS)n triplet repeat domain of rat dentin phosphophoryn.

Phosphophoryns (PPs) are unique aspartic acid and phosphoserine-rich proteins present in all species of dentin. Rat incisor odontoblast cDNA libraries contain messages encoding several acidic phosphorylated, serine-rich proteins. At least two of these share a common C-terminal domain coding region sequence. The polypeptide sequences in the N-terminal direction immediately adjacent to the conserved C-terminal domains of these two proteins (DMP2, DMP3) are distinctly different. In this domain, the DMP2 has extensive sequences of (DSS)n repeats with n as large as 24. DMP3 has fewer and shorter triplet sequences, n = 3, 4. The major rat incisor PPs (90-95 kDa) probably have the (DSS)n>>3. We propose that the name phosphophoryn be reserved for the extracellular matrix proteins with these extended repeats. DMPI, although strongly acidic, does not fit this category. If the S residues are phosphorylated and n > 3, conformational energy minimization computations show the (DSS)n sequence to assume a unique extended structure with parallel arrays of carboxylate and phosphate groups which may function as Ca2+ ion interaction edges. The phosphorylation of recombinant DMP2 C-terminal domain by various kinases has been examined. The repeat domains are not direct substrates for the CK2-like kinases but the kinases act in concert, so that the phosphorylation is hierarchical, apparently controlled by the presence of specific interruptions between the triplet domains.

Phosphorylation of the proteins of the extracellular matrix of mineralized tissues by casein kinase-like activity.

The extracellular matrix of the connective tissue contains non-collagenous proteins (NCP) which are acidic in character. The NCP of mineralizing systems (bone, dentin) differ from those of the non-mineralizing systems (skin, tendon) in that the mineralized tissue NCP are frequently phosphorylated. The phosphorylated proteins have been implicated in various aspects of the mineralization process. Thus, it is of interest to consider the mechanism and regulation of phosphorylation of the major matrix NCP. The majority of the phosphorylation takes place at Ser or Thr residues embedded within acidic sequences, and therefore are targets for casein kinase I (CK1) or casein kinase II (CK2)-like kinases. CK1 and CK2 are distantly related members of the protein kinase family. They are ubiquitous, constitutively active, second-messenger-independent kinases. CK1 is found in a variety of isoforms, all homologous to the alpha-subunit of the protein kinase family. It acts as a monomer. The active form of CK2 is a tetrameric holoenzyme, with 2 alpha catalytic subunits and 2 beta regulatory subunits. The CK2 alpha has activity alone, but the holoenzyme is four- to five-fold that activity. CK2 can use either ATP or GTP as the phosphate donor, but CK1 can use only ATP. The CK2 activity which phosphorylates the mineralized tissue NCP appears to be localized to membrane-associated cell fractions, and is present in the endoplasmic reticulum and Golgi compartments in osteoblasts, where phosphorylation of the secreted proteins appears to take place as co- and post-translational processes. Data indicate that both alpha and beta subunits of the membrane-associated CK2 are isoforms of the cytosolic CK2 in the same cells. The CK1 has not been specifically localized. Studies of dephosphorylated NCP such as phosphophoryn (PP) have shown that CK1 will not phosphorylate dephosphorylated dPP unless prior phosphorylation with CK2 has been carried out. In turn, CK2 activity may be initiated only after an initial phosphorylation of one of the messenger-dependent kinases. Thus, the phosphorylation reactions in mineralized tissues may be a tightly regulated hierarchical or sequential cascade of intracellular phosphorylation events.