Novel approaches for periodontal tissue engineering.

The periodontal complex involves the hard and soft tissues which support dentition, comprised of cementum, bone, and the periodontal ligament (PDL). Periodontitis, a prevalent infectious disease of the periodontium, threatens the integrity of these tissues and causes irreversible damage. Periodontal therapy aims to repair and ultimately regenerate these tissues toward preserving native dentition and improving the physiologic integration of dental implants. The PDL contains multipotent stem cells, which have a robust capacity to differentiate into various types of cells to form the PDL, cementum, and alveolar bone. Selection of appropriate growth factors and biomaterial matrices to facilitate periodontal regeneration are critical to recapitulate the physiologic organization and function of the periodontal complex. Herein, we discuss the current state of clinical periodontal regeneration including a review of FDA-approved growth factors. We will highlight advances in preclinical research toward identifying additional growth factors capable of robust repair and biomaterial matrices to augment regeneration similarly and synergistically, ultimately improving periodontal regeneration's predictability and long-term efficacy. This review should improve the readers' understanding of the molecular and cellular processes involving periodontal regeneration essential for designing comprehensive therapeutic approaches.

New paradigms in regenerative engineering: Emerging role of extracellular vesicles paired with instructive biomaterials.

Mesenchymal stem cells (MSCs) have long been regarded as critical components of regenerative medicine strategies, given their multipotency and persistence in a variety of tissues. Recently, the specific role of MSCs in mediating regenerative outcomes has been attributed (in part) to secreted factors from transplanted cells, namely extracellular vesicles. This viewpoint manuscript highlights the promise of cell-derived extracellular vesicles as agents of regeneration, enhanced by synergy with appropriate biomaterials platforms. Extracellular vesicles are a potentially interesting regenerative tool to enhance the synergy between MSCs and biomaterials. As a result, we believe these technologies will improve patient outcomes through efficient therapeutic strategies resulting in predictable patient outcomes.

Scaffold Pore Curvature Influences ΜSC Fate through Differential Cellular Organization and YAP/TAZ Activity.

Tissue engineering aims to repair, restore, and/or replace tissues in the human body as an alternative to grafts and prostheses. Biomaterial scaffolds can be utilized to provide a three-dimensional microenvironment to facilitate tissue regeneration. Previously, we reported that scaffold pore size influences vascularization and extracellular matrix composition both in vivo and in vitro, to ultimately influence tissue phenotype for regenerating cranial suture and bone tissues, which have markedly different tissue properties despite similar multipotent stem cell populations. To rationally design biomaterials for specific cell and tissue fate specification, it is critical to understand the molecular processes governed by cell-biomaterial interactions, which guide cell fate specification. Building on our previous work, in this report we investigated the hypothesis that scaffold pore curvature, the direct consequence of pore size, modulates the differentiation trajectory of mesenchymal stem cells (MSCs) through alterations in the cytoskeleton. First, we demonstrated that sufficiently small pores facilitate cell clustering in subcutaneous explants cultured in vivo, which we previously reported to demonstrate stem tissue phenotype both in vivo and in vitro. Based on this observation, we cultured cell-scaffold constructs in vitro to assess early time point interactions between cells and the matrix as a function of pore size. We demonstrate that principle curvature directly influences nuclear aspect and cell aggregation in vitro. Scaffold pores with a sufficiently low degree of principle curvature enables cell differentiation; pharmacologic inhibition of actin cytoskeleton polymerization in these scaffolds decreased differentiation, indicating a critical role of the cytoskeleton in transducing cues from the scaffold pore microenvironment to the cell nucleus. We fabricated a macropore model, which allows for three-dimensional confocal imaging and demonstrates that a higher principle curvature facilitates cell aggregation and the formation of a potentially protective niche within scaffold macropores which prevents MSC differentiation and retains their stemness. Sufficiently high principle curvature upregulates yes-associated protein (YAP) phosphorylation while decreased principle curvature downregulates YAP phosphorylation and increases YAP nuclear translocation with subsequent transcriptional activation towards an osteogenic differentiation fate. Finally, we demonstrate that the inhibition of the YAP/TAZ pathway causes a defect in differentiation, while YAP/TAZ activation causes premature differentiation in a curvature-dependent way when modulated by verteporfin (VP) and 1-oleyl-lysophosphatidic acid (LPA), respectively, confirming the critical role of biomaterials-mediated YAP/TAZ signaling in cell differentiation and fate specification. Our data support that the principle curvature of scaffold macropores is a critical design criterion which guides the differentiation trajectory of mesenchymal stem cells' scaffolds. Biomaterial-mediated regulation of YAP/TAZ may significantly contribute to influencing the regenerative outcomes of biomaterials-based tissue engineering strategies through their specific pore design.

Platform technologies for regenerative endodontics from multifunctional biomaterials to tooth-on-a-chip strategies.

OBJECTIVES: The aim of this review is to highlight recent progress in the field of biomaterials-mediated dental pulp tissue engineering. Specifically, we aim to underscore the critical design criteria of biomaterial platforms that are advantageous for pulp tissue engineering, discuss models for preclinical evaluation, and present new and innovative multifunctional strategies that hold promise for clinical translation. MATERIALS AND METHODS: The current article is a comprehensive overview of recent progress over the last 5 years. In detail, we surveyed the literature in regenerative pulp biology, including novel biologic and biomaterials approaches, and those that combined multiple strategies, towards more clinically relevant models. PubMed searches were performed using the keywords: "regenerative dentistry," "dental pulp regeneration," "regenerative endodontics," and "dental pulp therapy." RESULTS: Significant contributions to the field of regenerative dentistry have been made in the last 5 years, as evidenced by a significant body of publications. We chose exemplary studies that we believe are progressive towards clinically translatable solutions. We close this review with an outlook towards the future of pulp regeneration strategies and their clinical translation. CONCLUSIONS: Current clinical treatments lack functional and predictable pulp regeneration and are more focused on the treatment of the consequences of pulp exposure, rather than the restoration of healthy dental pulp. CLINICAL RELEVANCE: Clinically, there is great demand for bioinspired biomaterial strategies that are safe, efficacious, and easy to use, and clinicians are eager for their clinical translation. In particular, we place emphasis on strategies that combine favorable angiogenesis, mineralization, and functional tissue formation, while limiting immune reaction, risk of microbial infection, and pulp necrosis.

Rapamycin rescues BMP mediated midline craniosynostosis phenotype through reduction of mTOR signaling in a mouse model.

Craniosynostosis is defined as congenital premature fusion of one or more cranial sutures. While the genetic basis for about 30% of cases is known, the causative genes for the diverse presentations of the remainder of cases are unknown. The recently discovered cranial suture stem cell population affords an opportunity to identify early signaling pathways that contribute to craniosynostosis. We previously demonstrated that enhanced BMP signaling in neural crest cells (caA3 mutants) leads to premature cranial suture fusion resulting in midline craniosynostosis. Since enhanced mTOR signaling in neural crest cells leads to craniofacial bone lesions, we investigated the extent to which mTOR signaling is involved in the pathogenesis of BMP-mediated craniosynostosis by affecting the suture stem cell population. Our results demonstrate a loss of suture stem cells in the caA3 mutant mice by the newborn stage. We have found increased activation of mTOR signaling in caA3 mutant mice during embryonic stages, but not at the newborn stage. Our study demonstrated that inhibition of mTOR signaling via rapamycin in a time specific manner partially rescued the loss of the suture stem cell population. This study provides insight into how enhanced BMP signaling regulates suture stem cells via mTOR activation.

Infection-free rates and Sequelae predict factors in bone transportation for infected tibia: a systematic review and meta-analysis.

BACKGROUND: Tibia infected nonunion and chronic osteomyelitis are challenging clinical presentations. Bone transportation with external or hybrid fixators (combined external and internal fixators) is versatile to solve these problems. However, the infection-free rates of these fixator systems are unknown. Additionally, the prognosis factors for results of bone transportation are obscure. Therefore, this systematic review and meta-analysis was conducted to answer these questions. METHODS: A systematic review was conducted following the PRISMA-IPD guidelines. Relevant publications from January 1995 to September 2018 were compiled from Medline, Embase, and Cochrane. The infection-free rates of external and hybrid fixators were achieved by synthesizing aggregate data and individual participant data (IPD). IPD was analyzed by two-stage method with logistical regression to identify prognosis factors of sequelae. RESULTS: Twenty-two studies with 518 patients were identified, including 11 studies with 167 patients' IPD, and 11 studies with 351 patients' aggregate data. The infection-free rate of hybrid fixator group was 86% (95%CI: 79-94%), lower than that of external fixator which was 97% (95%CI: 95-98%,). The number of previous surgeries was found predict factor of bone union sequelae (p = 0.04) and function sequelae(p < 0.01); The external fixation time was found predict factor of function sequelae (p = 0.015). CONCLUSIONS: Hybrid fixators may be associated with a greater risk of infection-recurrence in the treatment of tibia infected nonunion and chronic osteomyelitis. The number of previous surgeries and external fixation time can be used as predictors of outcomes. Proper fixators and meticulously designed surgery are important to avoid unexpected operations and shorten external fixation time.

Synthetic Periodontal Guided Tissue Regeneration Membrane with Self-Assembling Biphasic Structure and Temperature-Sensitive Shape Maintenance.

Periodontal disease poses significant challenges to the long-term stability of oral health by destroying the supporting structures of teeth. Guided tissue regeneration techniques, particularly barrier membranes, enable local regeneration by providing an isolated, protected compartment for osseous wound healing while excluding epithelial tissue. Here, this study reports on a thermosensitive periodontal membrane (TSPM) technology designed to overcome the mechanical limitations of current membranes through a semi-interpenetrating network of high molecular weight poly(L-lactic acid) (PLLA) and in situ-polymerized mesh of poly(ε-caprolactone)diacrylate (PCL-DA), and poly lactide-co-glycolide diacrylate (PLGA-DA). An optimized composition allows facile reshaping at greater than 52 °C and rigid shape maintenance at physiological temperature. Its unique bilayer morphology is achieved through self-assembly and thermally-induced phase separation, resulting in distinct yet continuous smooth and nanofibrous compartments adequate for epithelial occlusion and regeneration. Incorporating PLGA-DA enhances the membrane's hydrophilicity and degradation properties, facilitating a more rapid and controlled degradation and therapeutic delivery. This study demonstrates its ability to promote local regeneration by serving as a barrier membrane and simultaneously as a scaffolding matrix in a rat orthotopic periodontal defect model. The TSPM outperformed a clinically available material (Epi-Guide) to facilitate robust alveolar bone and periodontal ligament regeneration at 4 and 8 weeks.

Integration of Melt Electrowritten Polymeric Scaffolds and Bioprinting for Epithelial Healing via Localized Periostin Delivery.

Management of skin injuries imposes a substantial financial burden on patients and hospitals, leading to diminished quality of life. Periostin (rhOSF), an extracellular matrix component, regulates cell function, including a proliferative healing phase, representing a key protein to promote wound healing. Despite its proven efficacy in vitro, there is a lack of scaffolds that facilitate the in situ delivery of rhOSF. In addition, there is a need for a scaffold to not only support cell growth, but also to resist the mechanical forces involved in wound healing. In this work, we synthesized rhOSF-loaded mesoporous nanoparticles (MSNs) and incorporated them into a cell-laden gelatin methacryloyl (GelMA) ink that was bioprinted into melt electrowritten poly(ε-caprolactone) (PCL) microfibrous (MF-PCL) meshes to develop mechanically competent constructs. Diffraction light scattering (DLS) analysis showed a narrow nanoparticle size distribution with an average size of 82.7 ± 13.2 nm. The rhOSF-loaded hydrogels showed a steady and controlled release of rhOSF over 16 days at a daily dose of ∼40 ng/mL. Compared with blank MSNs, the incorporation of rhOSF markedly augmented cell proliferation, underscoring its contribution to cellular performance. Our findings suggest a promising approach to address challenges such as prolonged healing, offering a potential solution for developing robust, biocompatible, and cell-laden grafts for burn wound healing applications.

Amino Acid-Based Poly(ester urea) Biodegradable Membrane for Guided Bone Regeneration.

Barrier membranes (BM) for guided bone regeneration (GBR) aim to support the osteogenic healing process of a defined bony defect by excluding epithelial (gingival) ingrowth and enabling osteoprogenitor and stem cells to proliferate and differentiate into bone tissue. Currently, the most widely used membranes for these approaches are collagen-derived, and there is a discrepancy in defining the optimal collagen membrane in terms of biocompatibility, strength, and degradation rates. Motivated by these clinical observations, we designed a collagen-free membrane based on l-valine-co-l-phenylalanine-poly(ester urea) (PEU) copolymer via electrospinning. Degradation and mechanical properties of these membranes were performed on as-spun and water-aged samples. Alveolar-bone-derived stem cells (AvBMSCs) were seeded on the PEU BM to assess their cell compatibility and osteogenic characteristics, including cell viability, attachment/spreading, proliferation, and mineralized tissue-associated gene expression. In vivo, PEU BMs were subcutaneously implanted in rats to evaluate their potential to cause inflammatory responses and facilitate angiogenesis. Finally, critical-size calvarial defects and a periodontal model were used to assess the regenerative capacity of the electrospun PEU BM compared to clinically available Cytoflex synthetic membranes. PEU BM demonstrated equal biocompatibility to Cytoflex with superior mechanical performance in strength and elasticity. Additionally, after 14 days, PEU BM exhibited a higher expression of BGLAP/osteocalcin and superior in vivo performance-less inflammation and increased CD31 and VWF expression over time. When placed in critical-sized defects in the calvaria of rats, the PEU BM led to robust bone formation with high expression of osteogenesis and angiogenesis markers. Moreover, our membrane enhanced alveolar bone and cementum regeneration in an established periodontal model after 8 weeks. We demonstrate that the PEU BM exhibits favorable clinical properties, including mechanical stability, cytocompatibility, and facilitated bone formation in vitro and in vivo. This highlights its suitability for GBR in periodontal and craniofacial bone defects.

Mechanobiology-informed biomaterial and tissue engineering strategies for influencing skeletal stem and progenitor cell fate.

Skeletal stem and progenitor cells (SSPCs) are the multi-potent, self-renewing cell lineages that form the hematopoietic environment and adventitial structures of the skeletal tissues. Skeletal tissues are responsible for a diverse range of physiological functions because of the extensive differentiation potential of SSPCs. The differentiation fates of SSPCs are shaped by the physical properties of their surrounding microenvironment and the mechanical loading forces exerted on them within the skeletal system. In this context, the present review first highlights important biomolecules involved with the mechanobiology of how SSPCs sense and transduce these physical signals. The review then shifts focus towards how the static and dynamic physical properties of microenvironments direct the biological fates of SSPCs, specifically within biomaterial and tissue engineering systems. Biomaterial constructs possess designable, quantifiable physical properties that enable the growth of cells in controlled physical environments both in-vitro and in-vivo. The utilization of biomaterials in tissue engineering systems provides a valuable platform for controllably directing the fates of SSPCs with physical signals as a tool for mechanobiology investigations and as a template for guiding skeletal tissue regeneration. It is paramount to study this mechanobiology and account for these mechanics-mediated behaviors to develop next-generation tissue engineering therapies that synergistically combine physical and chemical signals to direct cell fate. Ultimately, taking advantage of the evolved mechanobiology of SSPCs with customizable biomaterial constructs presents a powerful method to predictably guide bone and skeletal organ regeneration.

Methacrylated Gelatin as an On-Demand Injectable Vehicle for Drug Delivery in Dentistry.

Gelatin methacrylate (GelMA) is a biodegradable and biocompatible engineered material with significant promise for its applications in tissue engineering, drug delivery, and 3D bioprinting applications. Gelatin is functionalized with terminal methacrylate groups which allow for its photoinducible crosslinking, and thereby tunable properties. Photocrosslinking of GelMA solution in situ allows for fabrication of hydrogels to fit patient-specific defects. Given its favorable biologic properties, GelMA may be used as a carrier for bioactive substances necessary to induce regenerative phenotypes or augment healing, such as growth factors and biotherapeutics. Gelatin is cleaved by cell-secreted enzymes such that its degradation, and subsequently release of bioactive substances, is well-matched to tissue regeneration processes. GelMA may be mixed with a wide array of additives to enhance and improve the specificity of its biologic activity. Here, we present two protocols for novel fabrications and their uses as clinically relevant drug delivery systems. GelMA hydrogels provides a versatile platform for the development of injectable drug delivery therapeutics for broad applications in regenerative dental medicine.

Temperature-responsive PCL-PLLA nanofibrous tissue engineering scaffolds with memorized porous microstructure recovery.

Biomaterial scaffolds in tissue engineering facilitate tissue regeneration and integration with the host. Poor healing outcomes arise from lack of cell and tissue infiltration, and ill-fitting interfaces between matrices or grafts, resulting in fibrous tissue formation, inflammation, and resorption. Existing tissue engineering scaffolds struggle to recover from deformation to fit irregularly shaped defects encountered in clinical settings without compromising their mechanical properties and favorable internal architecture. This study introduces a synthetic biomaterial scaffold composed of high molecular weight poly (L-lactic acid) (PLLA) and an interpenetrating network of poly (ε-caprolactone) (PCL), in a composition aiming to address the need for conformal fitting synthetic matrices which retain and recover their advantageous morphologies. The scaffold, known as thermosensitive memorized microstructure (TS-MMS), forms nanofibrous materials with memorized microstructures capable of recovery after deformation, including macropores and nanofibers. TS-MMS nanofibers, with 50-500 nm diameters, are formed via thermally induced phase separation (TIPS) of PLLA after in situ polymerization of PCL-diacrylate. A critical partial-melting temperature of TS-MMS at 52°C enables bulk deformation above this temperature, while retaining the nanofibrous and macroporous structures upon cooling to 37°C. Incorporation of drug-loaded poly (lactide-co-glycolide) (PLGA) nanoparticles directly into TS-MMS nanofibers during fabrication allows sustained release of a model drug for up to 40 days. Subcutaneous implantation in vivo using LysM-Cre;td-Tomato; Col1eGFP mice demonstrates successful cellularization and integration of deformed/recovered TS-MMS materials, surpassing the limitations of deformed PLLA scaffolds, to facilitate cell and vasculature infiltration requisite for successful bone regeneration. Additionally we demonstrated a method for embedding controlled release vehicles directly into the scaffold nanofibers; controlled release of simvastatin enhances vascularization and tissue maturation. TS-MMS scaffolds offer promising improvements in clinical handling and performance compared to existing biomaterial scaffolds.

Next-generation biomaterials for dental pulp tissue immunomodulation.

OBJECTIVES: The current standard for treating irreversibly damaged dental pulp is root canal therapy, which involves complete removal and debridement of the pulp space and filling with an inert biomaterial. A regenerative approach to treating diseased dental pulp may allow for complete healing of the native tooth structure and enhance the long-term outcome of once-necrotic teeth. The aim of this paper is, therefore, to highlight the current state of dental pulp tissue engineering and immunomodulatory biomaterials properties, identifying exciting opportunities for their synergy in developing next-generation biomaterials-driven technologies. METHODS: An overview of the inflammatory process focusing on immune responses of the dental pulp, followed by periapical and periodontal tissue inflammation are elaborated. Then, the most recent advances in treating infection-induced inflammatory oral diseases, focusing on biocompatible materials with immunomodulatory properties are discussed. Of note, we highlight some of the most used modifications in biomaterials' surface, or content/drug incorporation focused on immunomodulation based on an extensive literature search over the last decade. RESULTS: We provide the readers with a critical summary of recent advances in immunomodulation related to pulpal, periapical, and periodontal diseases while bringing light to tissue engineering strategies focusing on healing and regenerating multiple tissue types. SIGNIFICANCE: Significant advances have been made in developing biomaterials that take advantage of the host's immune system to guide a specific regenerative outcome. Biomaterials that efficiently and predictably modulate cells in the dental pulp complex hold significant clinical promise for improving standards of care compared to endodontic root canal therapy.

A library of Rhodamine6G-based pH-sensitive fluorescent probes with versatile in vivo and in vitro applications.

Acidic pH is critical to the function of the gastrointestinal system, bone-resorbing osteoclasts, and the endolysosomal compartment of nearly every cell in the body. Non-invasive, real-time fluorescence imaging of acidic microenvironments represents a powerful tool for understanding normal cellular biology, defining mechanisms of disease, and monitoring for therapeutic response. While commercially available pH-sensitive fluorescent probes exist, several limitations hinder their widespread use and potential for biologic application. To address this need, we developed a novel library of pH-sensitive probes based on the highly photostable and water-soluble fluorescent molecule, Rhodamine 6G. We demonstrate versatility in terms of both pH sensitivity (i.e., pK a) and chemical functionality, allowing conjugation to small molecules, proteins, nanoparticles, and regenerative biomaterial scaffold matrices. Furthermore, we show preserved pH-sensitive fluorescence following a variety of forms of covalent functionalization and demonstrate three potential applications, both in vitro and in vivo, for intracellular and extracellular pH sensing. Finally, we develop a computation approach for predicting the pH sensitivity of R6G derivatives, which could be used to expand our library and generate probes with novel properties.

Macropore design of tissue engineering scaffolds regulates mesenchymal stem cell differentiation fate.

Craniosynostosis is a debilitating birth defect characterized by the premature fusion of cranial bones resulting from premature loss of stem cells located in suture tissue between growing bones. Mesenchymal stromal cells in long bone and the cranial suture are known to be multipotent cell sources in the appendicular skeleton and cranium, respectively. We are developing biomaterial constructs to maintain stemness of the cranial suture cell population towards an ultimate goal of diminishing craniosynostosis patient morbidity. Recent evidence suggests that physical features of synthetic tissue engineering scaffolds modulate cell and tissue fate. In this study, macroporous tissue engineering scaffolds with well-controlled spherical pores were fabricated by a sugar porogen template method. Cell-scaffold constructs were implanted subcutaneously in mice for up to eight weeks then assayed for mineralization, vascularization, extracellular matrix composition, and gene expression. Pore size differentially regulates cell fate, where sufficiently large pores provide an osteogenic niche adequate for bone formation, while sufficiently small pores (<125 µm in diameter) maintain stemness and prevent differentiation. Cell-scaffold constructs cultured in vitro followed the same pore size-controlled differentiation fate. We therefore attribute the differential cell and tissue fate to scaffold pore geometry. Scaffold pore size regulates mesenchymal cell fate, providing a novel design motif to control tissue regenerative processes and develop mesenchymal stem cell niches in vivo and in vitro through biophysical features.

Scaffolds with controlled release of pro-mineralization exosomes to promote craniofacial bone healing without cell transplantation.

Biomimetic bone regeneration methods which demonstrate both clinical and manufacturing feasibility, as alternatives to autogenic or allogenic bone grafting, remain a challenge to the field of tissue engineering. Here, we report the pro-osteogenic capacity of exosomes derived from human dental pulp stem cells (hDPSCs) to facilitate bone marrow stromal cell (BMSC) differentiation and mineralization. To support their delivery, we engineered a biodegradable polymer delivery platform to improve the encapsulation and the controlled release of exosomes on a tunable time scale from poly(lactic-co-glycolic acid) (PLGA) and poly(ethylene glycol) (PEG) triblock copolymer microspheres. Our delivery platform integrates within three-dimensional tissue engineering scaffolds to enable a straightforward surgical insertion into a mouse calvarial defect. We demonstrate the osteogenic potential of these functional constructs in vitro and in vivo. Controlled release of osteogenic hDPSC-derived exosomes facilitates osteogenic differentiation of BMSCs, leading to mineralization to a degree which is comparable to exogenous administration of the same exosomes in human and mouse BMSCs. By recruiting endogenous cells to the defects and facilitating their differentiation, the controlled release of osteogenic exosomes from a tissue engineering scaffold demonstrates accelerated bone healing in vivo at 8 weeks. Exosomes recapitulate the advantageous properties of mesenchymal stem/progenitor cells, without manufacturing or immunogenic concerns associated with transplantation of exogenous cells. This biomaterial platform enables exosome-mediated bone regeneration in an efficacious and clinically relevant way.

Controlled release of odontogenic exosomes from a biodegradable vehicle mediates dentinogenesis as a novel biomimetic pulp capping therapy.

Mineralized enamel and dentin provide protection to the dental pulp, which is vital tissue rich with cells, vasculature, and nerves in the inner tooth. Dental caries left untreated threaten exposure of the dental pulp, providing facile access for bacteria to cause severe infection both in the pulp and systemically. Dental materials which stimulate the formation of a protective dentin bridge after insult are necessary to seal the pulp chamber in an effort to maintain natural dentition and prevent pulpal infection. Dental materials to date including calcium hydroxide paste, mineral trioxide aggregate, and glass ionomer resin, are used with mixed results. Herein we exploited the cell-cell communicative properties of exosomes, extracellular vesicles derived from both mineralizing primary human dental pulp stem cells (hDPSCs) and an immortalized murine odontoblast cell line (MDPC-23), to catalyze the formation of a reactionary dentin bridge by recruiting endogenous stem cells of the dental pulp, through an easy-to-handle delivery vehicle which allows for their therapeutic controlled delivery at the pulp interface. Exosomes derived from both hDPSCs and MDPCs upregulated odontogenic gene expression and increased mineralization in vitro. We designed an amphiphilic synthetic polymeric vehicle from a triblock copolymer which encapsulates exosomes by polymeric self-assembly and maintains their biologic integrity throughout release up to 8-12 weeks. The controlled release of odontogenic exosomes resulted in a reparative dentin bridge formation, superior to glass-ionomer cement alone in vivo, in a rat molar pulpotomy model after six weeks. We have developed a platform for the encapsulation and controlled, tunable release of cell-derived exosomes, which maintains their advantageous physiologic properties reflective of the donor cells. This platform is used to modulate downstream recipient cells towards a designed dentinogenic trajectory in vitro and in vivo. Additionally, we have demonstrated the utility of an immortalized cell line to produce a high yield of exosomes with cross-species efficacy.

Three-Dimensional Electrodeposition of Calcium Phosphates on Porous Nanofibrous Scaffolds and Their Controlled Release of Calcium for Bone Regeneration.

To mimic the bone matrix of mineralized collagen and to impart microporous structure to facilitate cell migration and bone regeneration, we developed a nanofibrous (NF) polymer scaffold with highly interconnected pores and three-dimensional calcium phosphate coating utilizing an electrodeposition technique. The mineral content, morphology, crystal structure, and chemical composition could be tailored by adjusting the deposition temperature, voltage, and duration. A higher voltage and a higher temperature led to a greater rate of mineralization. Furthermore, nearly linear calcium releasing kinetics was achieved from the mineralized 3D scaffolds. The releasing rate was controlled by varying the initial electrodeposition conditions. A higher deposition voltage and temperature led to slower calcium release, which was associated with the highly crystalline and stoichiometric hydroxyapatite content. This premineralized NF scaffold enhanced bone regeneration over the control scaffold in a subcutaneous implantation model, which was associated with released calcium ions in facilitating osteogenic cell proliferation.

Pore size directs bone marrow stromal cell fate and tissue regeneration in nanofibrous macroporous scaffolds by mediating vascularization.

In the U.S., 30% of adults suffer joint pain, most commonly in the knee, which severely limits mobility and is often attributed to injury of cartilage and underlying bone in the joint. Current treatment methods such as microfracture result in less resilient fibrocartilage with eventual failure; autografting can cause donor site morbidity and poor integration. To overcome drawbacks in treatment, tissue engineers can design cell-instructive biomimetic scaffolds using biocompatible materials as alternate therapies for osteochondral defects. Nanofibrous poly (l-lactic acid) (PLLA) scaffolds of uniform, spherical, interconnected and well-defined pore sizes that are fabricated using a thermally-induced phase separation and sugar porogen template method create an extracellular matrix-like environment which facilitates cell adhesion and proliferation. Herein we report that chondrogenesis and endochondral ossification of rabbit and human bone marrow stromal cells (BMSCs) can be controlled by scaffold pore architecture, particularly pore size. Small-pore scaffolds support enhanced chondrogenic differentiation in vitro and cartilage formation in vivo compared to large-pore scaffolds. Endochondral ossification is prevented in scaffolds with very small pore sizes; pore interconnectivity is critical to promote capillary ingrowth for mature bone formation. These results provide a novel strategy to control tissue regenerative processes by tunable architecture of macroporous nanofibrous scaffolds. STATEMENT OF SIGNIFICANCE: Progress in understanding the relationship between cell fate and architectural features of tissue engineering scaffolds is critical for engineering physiologically functional tissues. Sugar porogen template scaffolds have uniform, spherical, highly interconnected macropores. Tunable pore-size guides the fate of bone marrow stromal cells (BMSCs) towards chondrogenesis and endochondral ossification, and is a critical design parameter to mediate neotissue vascularization. Preventing vascularization favors a chondrogenic cell fate while allowing vascularization results in endochondral ossification and mineralized bone formation. These results provide a novel strategy to control tissue regenerative processes by tunable architecture of macroporous nanofibrous scaffolds.