The temporomandibular joint (TMJ), essential for jaw movements, is susceptible to osteoarthritis, impacting a significant portion of the population. This study introduces an innovative genetic mouse model to explore TMJ development, maintenance, and interactions with the mechanical environment. We exploited Evc2/Limbin conditional KO (Evc2 cKO) mice, specifically targeting neural crest cell-derived tissues (Wnt1Cre), to observe TMJ development. Disruption of Evc2 in neural crest cells contributed to morphological changes in the TMJ growth plate cartilage layers, predisposing the joint components to defects. Condyle defective regions presented a unique environment composed of cartilage, bone, stem cells, and an augmented polymorphic layer. Our findings further revealed that the Evc2 cKO mice presented TMJ components degeneration clinically like those observed in human TMJ-osteoarthritis (OA). Mandible condyle gene expression analysis showed augmented expression of general inflammatory and OA markers. Supplying the mice with regular chow worsens the phenotype, but soft chow fed partially rescued both condyle morphology and intra-articular space. The data suggest that changes in the loading environment critically affect the integrity and functionality of the TMJ, with direct implications for its preservation and disease management.

Introduction: Fibroblast growth factor 2 (FGF2) plays a crucial role in regulating the osteogenic differentiation of progenitor cells. However, the process by which this occurs is not yet fully understood. In this study, we aimed to investigate whether FGF2 stimulates the osteogenesis of precursor cells through the yes-associated protein (YAP) and large tumor suppressor kinases 1/2 (LATS1/2). Methods: Human bone marrow stromal cells (hBMSCs) were cultured in osteogenic medium supplemented with FGF2 at concentrations of 2 ng/mL, 10 ng/mL, and 50 ng/mL for 2, 7, or 21 days. Alizarin red staining was performed to identify mineralization after 21 days of culture. RT-qPCR was conducted to detect the mRNA expression of Yap, Lats1, Lats2, Runx2, Bglap, and β-Actin. Immunofluorescence staining was carried out to detect the protein expression of YAP and LATS1/2. Data was analyzed with a p-value set at 0.05. Results: Mineralization was most significant at 10 ng/ml of FGF2 for 7 days and increased with concentrations of FGF2 from 0 ng/ml to 10 ng/ml for 7 days (p < 0.05) but decreased at the high concentration of 50 ng/ml for 2 days (p < 0.05). mRNA expression of Yap, Runx2, and Bglap increased in concordance with the increasing mineralization levels, but Lats1/2 mRNA decreased. mRNA expression levels were dose-dependent when FGF2 was added for 7 days (p < 0.05) and time-dependent when FGF2 concentration was at 10 ng/ml (p < 0.05). At the protein level, YAP increased while LATS1/2 decreased, indicating that LATS1/2 decreased, and YAP increased at higher mineralization levels when hBMSCs were cultured with 10 ng/ml of FGF2 for 7 days. Discussion: Consistent with our results, prior research has also indicated that lower concentrations of FGF2 enhance cell proliferation, thereby increasing the cell population for later osteogenic differentiation. However, excessive expansion can negatively affect differentiation. The mechanism of FGF2 regulation in stem cell osteogenic differentiation needs more exploration. Conclusion: Optimal concentrations and durations of FGF2 are critical for the osteogenic differentiation of hBMSCs. Moreover, it has been observed that mineralization correlates well with increasing YAP and decreasing LATS1/2 during osteogenic differentiation.

More than 500,000 bone grafting procedures are performed annually in the USA. Considering the significant limitations of available bone grafts, we previously invented a phase-separation technology to generate nanofibrous poly(l-lactic acid) (PLLA) scaffolds that mimic the bone matrix collagen in nanofiber geometry and enhance bone regeneration. Here we report the development of nanofibrous scaffolds with covalently attached synthetic peptides that mimic native collagen peptides to activate the two main collagen receptors in bone cells, discoidin domain receptor 2 (DDR2) and β1 integrins. We synthesized a PLLA-based graft-copolymer to enable covalent peptide conjugation via a click reaction. Using PLLA and the graft-copolymer, we developed 3D scaffolds with interconnected pores and peptides-containing nanofibers to activate DDR2 and β1 integrins of osteogenic cells. The degradation rate and mechanical properties of the scaffolds are tunable. The peptides-decorated nanofibrous scaffolds demonstrated 7.8 times more mineralized bone regeneration over the control scaffolds without the peptides in a critical-sized bone defect regeneration model after 8 weeks of implantation, showing a synergistic effect of the two peptides. This study demonstrates the power of scaffolds to mimic ECM at both nanometer and molecular levels, activating cell surface receptors to liberate the innate regenerative potential of host stem/progenitor cells.

Tissue engineering is a multidisciplinary field grown rapidly over the past few decades, whose success most often requires bioactive scaffolds to structurally support and biologically direct cell fate and tissue regeneration. Poly(l-lactic acid) (PLLA) is a widely used tissue engineering material with high strength, biocompatibility, biodegradability, and the capability of nanofiber formation through thermally induced phase separation (TIPS). To enable biomolecule conjugation, a new PLLA-based block copolymer, poly(spiro-lactic-co-lactic acid)-block-poly(l-lactic acid) or PSLA-b-PLLA, was developed in this study. The new polymer PSLA-b-PLLA could also form nanofibers through TIPS, and excitingly greatly improved the mechanical properties over PLLA nanofibers, with up to 1.2 times tensile modulus, 12.1 times strain at break, 2.1 times ultimate strength and 35.1 times toughness of PLLA, while being able to conjugate bioactive molecules covalently using click reactions. Its degradation rate was also accelerated to facilitate tissue regeneration. The tissue engineering potential of the new polymer scaffold was evaluated using a mouse critical-sized bone regeneration model, showing 3.6 times more vascularized bone volume regeneration when covalently conjugated with a bone-morphogenetic-protein-2-derived peptide. The block copolymer satisfies multiple criteria for tissue engineering with tunable mechanical properties, degradation rate, and conjugation densities. It can be utilized to impart specific biomolecular signals and mechanical properties potentially for various other tissue engineering applications.

The scaffold is a porous three-dimensional (3D) material that supports cell growth and tissue regeneration. Such 3D structures should be generated with simple techniques and nontoxic ingredients to mimic bio-environment and facilitate tissue regeneration. In this work, simple but powerful techniques are demonstrated for the fabrication of lamellar and honeycomb-mimic scaffolds with poly(L-lactic acid). The honeycomb-mimic scaffolds with tunable pore size ranging from 70 to 160 µm are fabricated by crystal needle-guided thermally induced phase separation in a directional freezing apparatus. The compressive modulus of the honeycomb-mimic scaffold is ≈4 times higher than that of scaffold with randomly oriented pore structure. The fabricated honeycomb-mimic scaffold exhibits a hierarchical structure from nanofibers to micro-/macro-tubular structures. Pre-osteoblast MC3T3-E1 cells cultured on the honeycomb-mimic nanofibrous scaffolds exhibit an enhanced osteoblastic phenotype, with elevated expression levels of osteogenic marker genes, than those on either porous lamellar scaffolds or porous scaffolds with randomly oriented pores. The advanced techniques for the fabrication of the honeycomb-mimic structure may potentially be used for a wide variety of advanced functional materials.

Prosthesis-induced pathological calcification is a significant challenge in biomaterial applications and is often associated with various reconstructive medical procedures. It is uncertain whether the fibrous extracellular matrix (ECM) adjacent to biomaterials directly triggers osteogenic trans-differentiation in nearby cells. To investigate this possibility, we engineered a heterogeneous polystyrene fibrous matrix (PSF) designed to mimic the ECM. Our findings revealed that the myoblasts grown on this PSF acquired osteogenic properties, resulting in mineralization both in vitro and in vivo. Transcriptomic analyses indicated a notable upregulation in the expression of the long noncoding RNA metastsis-associated lung adenocarcinoma transcript 1 (Malat1) in the C2C12 myoblasts cultured on PSF. Intriguingly, silencing Malat1 curtailed the PSF-induced mineralization and downregulated the expression of bone morphogenetic proteins (Bmps) and osteogenic markers. Further, we found that PSF prompted the activation of Yap1 signaling and epigenetic modifications in the Malat1 promoter, crucial for the expression of Malat1. These results indicate that the fibrous matrix adjacent to biomaterials can instigate Malat1 upregulation, subsequently driving osteogenic trans-differentiation in myoblasts and ectopic calcification through its transcriptional regulation of osteogenic genes, including Bmps. Our findings point to a novel therapeutic avenue for mitigating prosthesis-induced pathological calcification, heralding new possibilities in the field of biomaterial-based therapies.

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Bone and joint-related diseases, including osteoarthritis (OA), rheumatoid arthritis (RA), and bone tumors, pose significant health challenges due to their debilitating effects on the musculoskeletal system. 14-3-3 proteins, a family of conserved regulatory molecules, play a critical role in the pathology of these diseases. This review discusses the intricate structure and multifunctionality of 14-3-3 proteins, their regulation of signaling pathways, and their interactions with other proteins. We underscore the significance of 14-3-3 proteins in the regulation of osteoblasts, osteoclasts, chondrocytes, and bone remodeling, all key factors in the maintenance and dysfunction of bone and joint systems. Specific focus is directed toward elucidating the contribution of 14-3-3 proteins in the pathology of OA, RA, and bone malignancies, where dysregulated 14-3-3-mediated signaling cascades have been implicated in the disease processes. This review illuminates how the perturbation of 14-3-3 protein interactions can lead to the pathological manifestations observed in these disorders, including joint destruction and osteolytic activity. We highlight cutting-edge research that positions 14-3-3 proteins as potential biomarkers for disease progression and as innovative therapeutic targets, offering new avenues for disease intervention and management.

The current clinical applications of bone morphogenetic proteins (BMPs) are limited to only a few specific indications. Locally controlled delivery of combinations of growth factors can be a promising strategy to improve BMP-based bone repair. However, the success of this approach requires the development of an effective release system and the correct choice of growth factors capable of enhancing BMP activity. Basic fibroblast growth factor (bFGF, also known as FGF-2) has shown promise in promoting bone repair, although conflicting results have been reported. Considering the complex biological activities of FGF-2, we hypothesized that FGF-2 can promote BMP-induced bone regeneration only if the dosage and kinetic parameters of the two factors are individually tailored. In this study, we conducted systematic in vitro studies on cell proliferation, differentiation, and mineralization in response to factor dose, delivery mode (sequential or simultaneous), and release rate. Subsequently, we designed individually controlled BMP-7 and FGF-2 release poly(lactide-co-glycolide) (PLGA) nanospheres attached to the poly(l-lactic acid) (PLLA) nanofibrous scaffolds. The data showed that BMP-7-induced bone formation was accelerated by a relatively higher FGF-2 dose (100 ng/scaffold) delivered at a faster release rate, or by a relatively lower FGF-2 dose (10 ng/scaffold) at a slower release rate in an in vivo bone regeneration model. In contrast, a very high dose of FGF-2 (1000 ng/scaffold) inhibited bone regeneration under all conditions. In vitro and in vivo data suggest that FGF-2 improved BMP-7-induced bone regeneration by coordinating FGF-2 dosage and release kinetics to enhance stem cell migration, proliferation, and angiogenesis. Statement of significance Bone morphogenetic proteins (BMPs) are the most potent growth/differentiation factors in bone development and regeneration. However, the clinical applications of BMPs have been limited to only a few specific indications due to the required supraphysiological dosages with the current BMP products and their side effects. Locally controlled delivery of BMPs and additional growth factors that can enhance their osteogenic potency are highly desired. However, different growth factors act with different mechanisms. Here we report a nanofibrous scaffold that mimics collagen in size and geometry and is immobilized with biodegradable nanospheres to achieve local and distinct release profiles of BMP7 and FGF2. Systematic studies demonstrated low dose BMP7 and FGF2 with different temporal release profiles can optimally enhance bone regeneration.

Stimuli-responsive domains capable of releasing loaded molecules, "on-demand," have garnered increasing attention due to their enhanced delivery, precision targeting, and decreased adverse effects. The development of an on-demand delivery system that can be easily triggered by dental clinicians might have major roles in dental and oral tissue engineering. A series of random graft poly(NIPAm-co-HEMA-Lactate) copolymers were synthesized using 95:5, 85:5, 60:40, and 40:60 ratios of thermosensitive NIPAm and HEMA-poly lactate respectively then electrospun to produce nanofibrous scaffolds loaded with bovine serum albumin (BSA). Cumulative BSA release was assessed at 25C and 37°C. To appraise the use of scaffolds as on-demand delivery systems, they were subjected to thermal changes in the form cooling and warming cycles during which BSA release was monitored. To confirm the triggered releasing ability of the synthesized scaffolds, the copolymer made with 60% NIPAm was selected, based on the results of the release tests, and loaded with bone morphogenetic protein-2 (BMP-2). The loaded scaffolds were placed with mesenchymal-like stem cells (iMSCs) derived from induced pluripotent stem cells (iPSCs), and subjected to temperature alterations. Then, the osteogenic differentiation of iMSCs, which might have resulted from the released protein, was evaluated after 10 days by analyzing runt-related transcription factor 2 (RUNX-2) osteogenic gene expression by the cells using real-time quantitative polymerase chain reaction (qRT-PCR). BSA release profiles showed a burst release at the beginning followed by a more linear pattern at 25°C, and a much slower release at 37°C. The release also decreased when the PNIPAm content decreased in the scaffolds. Thermal triggering led to a step-like release pattern in which the highest release was reported 30 min through the warming cycles. The iMSCs cultivated with scaffolds loaded with BMP-2 and exposed to temperature alteration showed significantly higher RUNX-2 gene expression than cells in the other experimental groups. The synthesized scaffolds are thermo-responsive and could be triggered to deliver biological biomolecules to be used in oral and dental tissue engineering. Thermal stimuli could be simulated by dental clinicians using simple means of cold therapy, for example, cold packs in intraoral accessible sites for specified times.

Xylitol is a five-carbon sugar alcohol, i.e. polyol that has beneficial health properties such as noncariogenicity and a preventive agent against osteoporosis by increasing bone density. However, its potential to induce odontogenic differentiation on dental pulp stem cells (DPSCs) remains unclear. Thus, this study aimed to investigate the capacity of xylitol to induce odontogenic differentiation and mineralization of DPSCs. Dental pulp stem cells (DPSCs) were isolated from human third molars and cultured in complete medium (CM) Minimum Essential Medium Eagle—Alpha Modification (α-MEM) with 10% fetal bovine serum, 100 units/mL penicillin, and 100 μg/mL streptomycin through several successive subcultures. The cells from passage 3 were used for the entire experiment. To evaluate the effect of xylitol on odontogenic differentiation, DPSCs were treated with 0.5 mM, 1 mM or 10 mM Xylitol in odontogenic medium containing CM with 50 μg/mL ascorbic acid, 5 mM β-glycerol phosphate, and 10−7 M dexamethasone. Control group was treated with odontogenic medium only (OSs) Cell viability assay was used to detect the cytotoxicity of xylitol on DPSCs and was determined by 2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Odontogenic activity and mineralization of DPSCs was assessed by alkaline phosphatase (ALP) assay and alizarin red staining (ARS), respectively. Odontogenic differentiation of DPSCs was evaluated by real-time quantitative polymerase chain reaction (RT–qPCR) for odontogenic markers: dentin sialophosphoprotein (DSPP), dentin matrix protein 1 (DMP1), alkaline phosphatase (ALP) and collagen type I (COL1), on day 7 and 14 which were normalized to the house keeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Untreated DPSCs were used as a control throughout the study. Data were analyzed with one-way ANOVA at α=0.05 Cells treated with xylitol at all concentrations tested maintained viability and there was no statistically significant difference in cell viability between the control and xylitol-treated groups, indicating that used concentration of xylitol were biocompatible (p>0.05). DPSCs treated with 1 mM xylitol showed the highest ALP activity (p< 0.05). The amount of culture mineralization and formation of mineralized nodules on DPSCs was higher for the group treated with 1 mM xylitol compared to control. These findings suggest that Xylitol induces odontogenic differentiation and mineralization of DPSCs.

Gene therapy and gene delivery have drawn extensive attention in recent years especially when the COVID-19 mRNA vaccines were developed to prevent severe symptoms caused by the corona virus. Delivering genes, such as DNA and RNA into cells, is the crucial step for successful gene therapy and remains a bottleneck. To address this issue, vehicles (vectors) that can load and deliver genes into cells are developed, including viral and non-viral vectors. Although viral gene vectors have considerable transfection efficiency and lipid-based gene vectors become popular since the application of COVID-19 vaccines, their potential issues including immunologic and biological safety concerns limited their applications. Alternatively, polymeric gene vectors are safer, cheaper, and more versatile compared to viral and lipid-based vectors. In recent years, various polymeric gene vectors with well-designed molecules were developed, achieving either high transfection efficiency or showing advantages in certain applications. In this review, we summarize the recent progress in polymeric gene vectors including the transfection mechanisms, molecular designs, and biomedical applications. Commercially available polymeric gene vectors/reagents are also introduced. Researchers in this field have never stopped seeking safe and efficient polymeric gene vectors via rational molecular designs and biomedical evaluations. The achievements in recent years have significantly accelerated the progress of polymeric gene vectors toward clinical applications.

The aim of this study was to investigate the effect of short-term (3 days) preconditioning with pro-inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) on proliferation and odontogenic differentiation of stem cells of apical papilla (SCAP) seeded on a 3D scaffold. Our results showed that while TNF-alpha preconditioning at various concentrations did not affect SCAP proliferation, it enhanced alkaline phosphatase (ALP) activity and mineralized matrix deposition in culture. Preconditioning SCAP using TNF-alpha at a concentration of about 1 ng/ml optimally enhanced odontogenic gene expression of SCAP in culture. Highly porous nanofibrous (NF) poly(L- lactic acid) (PLLA) scaffolds, with nanofiber features mimicking the environment of the extracellular matrix, were fabricated using a thermally induced phase separation method. TNF-alpha preconditioning of SCAP at 1 ng/ml promoted ALP activity, calcium deposition and mineralized tissue formation of SCAP cultured on 3D NF-PLLA scaffolds in vitro. Moreover, TNF-alpha preconditioning at a concentration of 1 ng/ml promoted the formation of more mineralized tissue as well as stronger DSPP expression of SCAP seeded on NF-PLLA scaffolds in vivo. Collectively, this study suggests that short term TNF-alpha preconditioning, mimicking the transiently low concentration TNF-alpha present during inflammation, may serve as a feasible approach to enhance the odontogenic differentiation of stem/progenitor cells on a biomimetic scaffold for 3D dentin regeneration. These results present a first step in the development of a dental tissue engineering strategy where odontogenesis is enhanced by modulating inflammation.

Skeletal progenitor: collagen interactions are critical for bone development and regeneration. Both collagen-binding integrins and discoidin domain receptors (DDR1 and DDR2) function as collagen receptors in bone. Each receptor is activated by a distinct collagen sequence; GFOGER for integrins and GVMGFO for DDRs. Specific triple helical peptides containing each of these binding domains were evaluated for ability to stimulate DDR2 and integrin signaling and osteoblast differentiation. GVMGFO peptide stimulated DDR2 Y740 phosphorylation and osteoblast differentiation as measured by induction of osteoblast marker mRNAs and mineralization without affecting integrin activity. In contrast, GFOGER peptide stimulated focal adhesion kinase (FAK) Y397 phosphorylation, an early measure of integrin activation, and to a lesser extent osteoblast differentiation without affecting DDR2-P. Significantly, the combination of both peptides cooperatively enhanced both DDR2 and FAK signaling and osteoblast differentiation, a response that was blocked in Ddr2-deficient cells. These studies suggest that the development of scaffolds containing DDR and integrin-activating peptides may provide a new route for promoting bone regeneration.

Sulfated polysaccharides of red marine microalgae have recently gained much attention for biomedical applications due to their anti-inflammatory and antioxidant properties. However, their low mechanical properties limit their use in tissue engineering. Herein, to enhance the mechanical properties of the sulfated polysaccharide produced by the red marine microalga, Porphyridium sp. (PS), it was integrated with the fluorenylmethoxycarbonyl diphenylalanine (FmocFF) peptide hydrogelator. Transparent, stable hydrogels were formed when mixing the two components at a 1:1 ratio in three different concentrations. Electron microscopy showed that all hydrogels exhibited a nanofibrous structure, mimicking the extracellular matrix. Furthermore, the hydrogels were injectable, and tunable mechanical properties were obtained by changing the hydrogel concentration. The composite hydrogels allowed the sustained release of curcumin which was controlled by the change in the hydrogel concentration. Finally, the hydrogels supported MC3T3-E1 preosteoblasts viability and calcium deposition. The synergy between the sulfated polysaccharide, with its unique bioactivities, and FmocFF peptide, with its structural and mechanical properties, bears a promising potential for developing novel tunable scaffolds for tissue engineering that may allow cell differentiation into various lineages.

Polymeric scaffolds play a pivotal role in tissue engineering. This chapter will focus on the state-of-the-art polymeric scaffolds and exciting recent advancements. First, the general concepts and requirements of tissue engineering biomaterials are introduced. Second, various types of polymeric biomaterials used in tissue engineering are reviewed. Third, important fabrication technologies of 3D scaffolds and their structural characteristics are discussed. Fourth, surface modification and composite scaffold fabrication techniques are presented as ways to improve scaffold properties. Then advanced nanostructured polymer scaffolds with biomolecule-delivering capacity are highlighted. Finally, the future perspectives of polymeric scaffolds for tissue engineering are discussed.

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.