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.

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.

Guided tissue regeneration (GTR), which is based on creating a physical barrier to prevent the downgrowth of epithelial and connective tissues into the defect site, has been widely used in clinical practice for periodontal regeneration for many years. However, its outcomes remain variable due to highly specific indications, the demand for proficient surgical skills, and frequent occurrence of complications. In this study, we developed a new GTR biomaterial that acts as a biological barrier for epithelial cells and fibroblasts while also serving as a scaffold for bone marrow-derived mesenchymal stem cells (BMSCs) and periodontal ligament stem cells (PDLSCs). This innovative GTR biomaterial is bioinspired injectable microspheres that are self-assembled from nanofibers, and their surfaces are conjugated with E7, a short peptide that selectively promotes BMSC and PDLSC adhesion but inhibits the attachment and spreading of epithelial cells and gingival fibroblasts. The selective affinity afforded by E7 on the surfaces of the nanofibrous microspheres facilitated the colonization of BMSCs in the periodontal defect, thereby substantially improving functional periodontal regeneration, as evidenced by enhanced new bone formation, reduced root exposure, and diminished attachment loss. The remarkable superiority of the bioinspired microspheres over conventional GTR materials in promoting periodontal regeneration underscores the potential of this innovative approach to enhance the efficacy of functional periodontal tissue regeneration.

Pickering emulsion polymerization, stabilized by inorganic nanoparticles such as iron oxide nanoparticles (IONPs), can be used to fabricate scaffolds with the desired porosity and pore size. These nanoparticles create stable emulsions that can be processed under harsh polymerization conditions. IONPs, apart from serving as an emulsifier, impart beneficial bioactivities such as antibacterial and pro-angiogenic activity. Here, we coated IONPs with three different weights of oleic acid (5.0 g, 7.5 g, and 10.0 g) to synthesize oleic acid-IONPs (OA-IONPs) that possess the desired hydrophobicity (contact angle > 100°). Next, glycidyl methacrylate and trimethylolpropane triacrylate were polymerized using the Pickering emulsion polymerization technique stabilized by the OA-IONPs. The physicochemical properties of the resulting porous scaffolds were thoroughly characterized using scanning electron microscopy (SEM), thermogravimetric analysis (TGA), Fourier transform infrared spectroscopy (FT-IR), vibrating sample magnetometry (VSM), and a universal testing machine (UTM). The SEM images confirmed the formation of a porous scaffold. The IONPs content, measured using inductively coupled plasma mass spectrometry (ICP-MS), was in the range of 22-26 µg/mg of the scaffold. The mechanical strengths of the scaffolds were in the range of cancellous bone. The degradation profile of the scaffolds varied between 29% and 41% degradation over 30 days. In vitro cytotoxicity studies conducted using the fibroblast (L929) and osteosarcoma (MG-63) cell lines proved that these scaffolds were non-toxic. SEM images showed that the MG-63 cells adhered firmly to the scaffolds and exhibited a well-spread morphology. The antibacterial activity was confirmed by percentage inhibition studies, SEM analysis of bacterial membrane distortion, and reactive oxygen species (ROS) generation in the bacteria. Chick chorioallantoic membrane assay showed that the total vessel length and branch points were significantly increased in the presence of the scaffolds. These results confirm the pro-angiogenic potential of the fabricated scaffolds. The physicochemical, mechanical, and biological properties of the material suggest that the developed scaffolds would be suitable for bone tissue engineering applications.

In the present study, extracellular matrix (ECM)-mimicking nanofibrous chitosan microspheres (NCM) were developed via thermal induction of chitosan molecular chain from alkaline/urea aqueous solution. The regeneration of NCM from chitosan was proved to be physical process. The morphology of NCM could be precisely controlled by adjusting the initial solution concentration and the reaction temperature. The NCM possessed desirable in vitro/vivo biocompatibility and biodegradability. The excellent cell adhesion capability of NCM facilitated the formation of large-sized 3D geometric constructs in vitro. The NCM promoted in vitro osteogenic differentiation of rat bone marrow stem cells (rMSCs). Finally, pre-differentiated rMSCs-NCM constructs obviously enhanced in vivo bone healing of rat calvarial defects. This work opened up a new avenue for the construction of chitosan microspheres with ECM-like nanofibrous structure, indicated the great potential of the NCM as micro-Noah's Ark for stem cells to anchor, proliferate, and pre-differentiate for tissue engineering.

Human pluripotent stem cells (hPSCs) may be differentiated into any adult cell type and therefore hold incredible promise for cell therapeutics and disease modeling. There is increasing interest in three-dimensional (3D) hPSC culture because of improved differentiation outcomes and potential for scale up. Our team has recently described bioactive heparin (Hep)-containing core-shell microcapsules that promote rapid aggregation of stem cells into spheroids and may also be loaded with growth factors for the local and sustained delivery to the encapsulated cells. In this study, we explored the possibility of further modulating bioactivity of microcapsules through the use of an ultrathin coating composed of tannic acid (TA). Deposition of the TA film onto model substrates functionalized with Hep and poly(ethylene glycol) was characterized by ellipsometry and atomic force microscopy. Furthermore, the presence of the TA coating was observed to increase the amount of basic fibroblast growth factor (bFGF) incorporation by up to twofold and to extend its release from 5 to 7 days. Most significantly, TA-microcapsules loaded with bFGF induced higher levels of pluripotency expression compared to uncoated microcapsules containing bFGF. Engineered microcapsules described here represent a new stem cell culture approach that enables 3D cultivation and relies on local delivery of inductive cues.

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.

Biomaterials and tissue regeneration represent two fields of intense research and rapid advancement. Their combination allowed the utilization of the different characteristics of biomaterials to enhance the expansion of stem cells or their differentiation into various lineages. Furthermore, the use of biomaterials in tissue regeneration would help in the creation of larger tissue constructs that can allow for significant clinical application. Several studies investigated the role of one or more biomaterial on stem cell characteristics or their differentiation potential into a certain target. In order to achieve real advancement in the field of stem cell-based tissue regeneration, a careful analysis of the currently published information is critically needed. This review describes the fundamental description of biomaterials as well as their classification according to their source, bioactivity and different biological effects. The effect of different biomaterials on stem cell expansion and differentiation into the primarily studied lineages was further discussed. In conclusion, biomaterials should be considered as an essential component of stem cell differentiation strategies. An intense investigation is still required. Establishing a consortium of stem cell biologists and biomaterial developers would help in a systematic development of this field.

The ultimate goal of periodontal treatment is to regenerate the lost periodontal tissues. The interest in nanomaterials in dentistry is growing rapidly and has focused on improvements in various biomedical applications, such as periodontal regeneration and periodontal tissue engineering. To enhance periodontal tissue regeneration, hydroxyapatite (HA) was used in conjunction with other scaffold materials, such as Poly lactic-co-glycolic-acid (PLGA) and collagen (C). The main target of this study was to compare the effects of nano and macrostructures of the tissue scaffolds on cell behavior in vitro for periodontal tissue engineering.

Nanofibrillar and macroporous-spongious composite tissue scaffolds were produced using PLGA/C/HA. Subgroups with BMP-2 signal molecule and without HA were also created. The scaffolds were characterized by FTIR, SEM/EDX techniques, and mechanical tests. The scaffolds were compared in the periodontal ligament (PDL) and MCT3-E1 cell cultures. The cell behaviors; adhesions by SEM, proliferation by WST-1, differentiation by ALP and mineralization with Alizarin Red Tests were determined.

Cell adhesion and mineralization were higher in the nanofibrillar scaffolds compared to the macroporous-spongious scaffolds. Macroporous-spongious scaffolds seemed better for the proliferation of PDL cells and differentiation of MC3T3-E1-preosteoblastic cells, while nanofibrillar scaffolds were more convenient for the differentiation of PDL cells and proliferation of MC3T3-E1-preosteoblastic cells.

In general, nanofibrillar scaffolds showed more favorable results in cell behaviors, compared to the macroporous-spongious scaffolds, and mostly, BMP-2 and HA promoted the activities of the cells.

The complex interaction of cells with biomaterials (i.e., materiobiology) plays an increasingly pivotal role in the development of novel implants, biomedical devices, and tissue engineering scaffolds to treat diseases, aid in the restoration of bodily functions, construct healthy tissues, or regenerate diseased ones. However, the conventional approaches are incapable of screening the huge amount of potential material parameter combinations to identify the optimal cell responses and involve a combination of serendipity and many series of trial-and-error experiments. For advanced tissue engineering and regenerative medicine, highly efficient and complex bioanalysis platforms are expected to explore the complex interaction of cells with biomaterials using combinatorial approaches that offer desired complex microenvironments during healing, development, and homeostasis. In this review, we first introduce materiobiology and its high-throughput screening (HTS). Then we present an in-depth of the recent progress of 2D/3D HTS platforms (i.e., gradient and microarray) in the principle, preparation, screening for materiobiology, and combination with other advanced technologies. The Compendium for Biomaterial Transcriptomics and high content imaging, computational simulations, and their translation toward commercial and clinical uses are highlighted. In the final section, current challenges and future perspectives are discussed. High-throughput experimentation within the field of materiobiology enables the elucidation of the relationships between biomaterial properties and biological behavior and thereby serves as a potential tool for accelerating the development of high-performance biomaterials.

In vivo, stem cells reside in a three-dimensional (3D) extracellular microenvironment in which complicated biophysical and biochemical factors regulate their behaviors. Biomimicking of the stem cell-matrix interactions is an ideal approach for controlling the stem cell fate. This study investigates the effects of the incorporation of cell-adhesive ligands in 3D self-assembling peptide hydrogels to modulate stem cell survival, proliferation, maintenance of stemness, and osteogenic differentiation. The results show that the composite hydrogels were non-cytotoxic and effective for maintaining human amniotic mesenchymal stem cell (hAMSC) survival, proliferation and phenotypic characterization. The expression levels of pluripotent markers were also upregulated in the composite hydrogels. Under inductive media conditions, mineral deposition and mRNA expression levels of osteogenic genes of hAMSCs were enhanced. The increasing expression of integrin α- and β-subunits for hAMSCs indicates that the ligand-integrin interactions may modulate the cell fate for hAMSCs in composite hydrogels.

Bone is composed of dense and solid cortical bone and honeycomb-like trabecular bone. Although cortical bone provides the majority of mechanical strength for a bone, there are few studies focusing on cortical bone repair or regeneration. Osteons (the Haversian system) form structural and functional units of cortical bone. In recent years, emerging evidences have shown that the osteon structure (including osteocytes, lamellae, lacunocanalicular network, and Haversian canals) plays critical roles in bone mechanics and turnover. Therefore, reconstruction of the osteon structure is crucial for cortical bone regeneration. This article provides a systematic summary of recent advances in osteons, including the structure, function, turnover, and regenerative strategies. First, the hierarchical structure of osteons is illustrated and the critical functions of osteons in bone dynamics are introduced. Next, the modeling and remodeling processes of osteons at a cellular level and the turnover of osteons in response to mechanical loading and aging are emphasized. Furthermore, several bioengineering approaches that were recently developed to recapitulate the osteon structure are highlighted. Impact statement This review provides a comprehensive summary of recent advances in osteons, especially the roles in bone formation, remodeling, and regeneration. Besides introducing the hierarchical structure and critical functions of osteons, we elucidate the modeling and remodeling of osteons at a cellular level. Specifically, we highlight the bioengineering approaches that were recently developed to mimic the hierarchical structure of osteons. We expect that this review will provide informative insights and attract increasing attentions in orthopedic community, shedding light on cortical bone regeneration in the future.

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.

The mechanical properties of biologic scaffolds are critical to cellular interactions and hence functional response within the body. In the case of scaffolds for bone tissue regeneration, engineered scaffolds created by combining collagen with inorganic mineral are increasingly being explored, due to their favourable structural and chemical characteristics. Development of a method for controlling the mechanics of these scaffolds could lead to significant additional advantages by harnessing the intrinsic mechnotransduction pathways of stem cells via appropriate control of scaffold mechanical properties. Here we present a method for controlling the macroscale flexural modulus of mineralized collagen sheets, and the radial indentation modulus of the sheets' constituent collagen fibrils. Scaffolds were created starting with sheets of highly aligned, natively structured collagen fibrils, prepared via cryosectioning of decellularized tendon. Sheets underwent an alternate soaking mineralization procedure, with sequential exposure to citrate-doped calcium and carbonate-containing phosphate solutions, both of which included poly aspartic acid. The extent of scaffold mineralization was controlled via number of repeated mineralization cycles: 0 (unmineralized), 5, 10, and 20 cycles were trialed. Following scaffold preparation, ultrastructure, macroscale flexural modulus, and nanoscale indentation modulus were assessed. Surface architecture studied by SEM, and inspection of individual extracted fibrils by TEM and AFM confirmed that fibrils became increasingly laden with mineral as the number of mineralization cycles increased. Measurements of collagen fibril nanomechanics using AFM showed that the radial modulus of collagen fibrils increased linearly with mineralization cycles completed, from 215 ± 125 MPa for fibrils from unmineralized (0 cycle) scaffolds to 778 ± 302 MPa for fibrils from the 20 mineralization cycle scaffolds. Measurements of scaffold macromechanics via flexural testing also showed a linear increase in flexural modulus with increasing number of mineralization cycles completed, from 18 ± 7 MPa for the 5 cycle scaffolds to 156 ± 50 MPa for the 20 cycle scaffolds. The process detailed herein provides a way to create mineralized collagen scaffolds with easily controllable mechanical properties.

Bacteria use flagella, the protein nanofibers on their surface, as a molecular machine to swim. Flagella are polymerized from monomers, flagellins, which can display a peptide by genetic means. However, flagella as genetically modifiable nanofibers have not been used in building bone extracellular matrix-like structures for inducing stem cell differentiation in non-osteogenic medium. Here we discovered that interactions between Ca2+ ions and flagella (displaying a collagen-like peptide (GPP)8 on every flagellin) resulted in ordered bundle-like structures, which were further mineralized with hydroxyapatite to form ordered fibrous matrix. The resultant matrix significantly induced the osteogenic differentiation of stem cells, much more efficiently than wild-type flagella and type I collagen. This work shows that flagella can be used as protein building blocks in generating biomimetic materials.

Calcium phosphate cements (CPCs) mimic nanostructured bone minerals and are promising for dental, craniofacial and orthopedic applications. Vascularization plays a critical role in bone regeneration. This article represents the first review on cutting-edge research on prevascularization of CPC scaffolds to enhance bone regeneration.

This article first presented the prevascularization of CPC scaffolds. Then the co-culture of two cell types in CPC scaffolds was discussed. Subsequently, to further enhance the prevascularization efficacy, tri-culture of three different cell types in CPC scaffolds was presented.

(1) Arg-Gly-Asp (RGD) incorporation in CPC bone cement scaffold greatly enhanced cell affinity and bone prevascularization; (2) By introducing endothelial cells into the culture of osteogenic cells (co-culture of two different cell types, or bi-culture) in CPC scaffold, the bone defect area underwent much better angiogenic and osteogenic processes when compared to mono-culture; (3) Tri-culture with an additional cell type of perivascular cells (such as pericytes) resulted in a substantially enhanced prevascularization of CPC scaffolds in vitro and more new bone and blood vessels in vivo, compared to bi-culture. Furthermore, biological cell crosstalk and capillary-like structure formation made critical contributions to the bi-culture system. In addition, the pericytes in the tri-culture system substantially promoted stability and maturation of the primary vascular network.

The novel approach of CPC scaffolds with stem cell bi-culture and tri-culture is of great significance in the regeneration of dental, craniofacial and orthopedic defects in clinical practice.

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.

Clinical use of human embryonic stem cells (hESCs) in bone regeneration applications requires that their osteogenic differentiation be highly controllable as well as time- and cost-effective. The main goals of the current work were thus (a) to assess whether overexpression of pluripotency regulator Forkhead Box D3 (FOXD3) can enhance the osteogenic commitment of hESCs seeded in three-dimensional (3D) scaffolds and (b) to evaluate if the degree of FOXD3 overexpression regulates the strength and specificity of hESC osteogenic commitment. In conducting these studies, an interpenetrating hydrogel network consisting of poly(ethylene glycol) diacrylate and collagen I was utilized as a 3D culture platform. Expression of osteogenic, chondrogenic, pluripotency, and germ layer markers by encapsulated hESCs was measured after 2 weeks of culture in osteogenic medium in the presence or absence doxycycline-induced FOXD3 transgene expression. Towards the first goal, FOXD3 overexpression initiated 24 hr prior to hESC encapsulation, relative to unstimulated controls, resulted in upregulation of osteogenic markers and enhanced calcium deposition, without promoting off-target effects. However, when initiation of FOXD3 overexpression was increased from 24 to 48 hr prior to encapsulation, hESC osteogenic commitment was not further enhanced and off-target effects were noted. Specifically, relative to 24-hr prestimulation, initiation of FOXD3 overexpression 48 hr prior to encapsulation yielded increased expression of pluripotency markers while reducing mesodermal but increasing endodermal germ layer marker expression. Combined, the current results indicate that the controlled overexpression of FOXD3 warrants further investigation as a mechanism to guide enhanced hESC osteogenic commitment.

In addition to T cells' roles in immune response and autoimmune diseases, certain types of T cells, called regulatory T cells (Tregs), play important roles in microenvironment modulation for resolution and tissue regeneration. However, there are currently few options available other than introducing more Tregs or immunosuppressive drugs to locally enrich Tregs. Herein, poly(l-lactic acid) (PLLA) nanofibrous spongy microspheres (NF-SMS), PLLA/polyethylene glycol (PEG) co-functionalized mesoporous silica nanoparticles (MSN), and poly(lactic acid- co-glycolic acid) microspheres (PLGA MS) are integrated into one multibiologic delivery vehicle for in situ Treg manipulation, where the MSNs and PLGA MS were utilized to distinctly release IL-2/TGF-β and miR-10a to locally recruit T cells and stimulate their differentiation into Tregs, while PLLA NF-SMS serve as an injectable scaffold for the adhesion and proliferation of these Tregs. In a mouse model of periodontitis, the injectable and biomolecule-delivering PLLA NF-SMS lead to Treg enrichment, expansion, and Treg-mediated immune therapy against bone loss. This system can potentially be utilized in a wide variety of other immune and regenerative therapies.

Bone is the second most commonly transplanted tissue worldwide, with over four million operations using bone grafts or bone substitute materials annually to treat bone defects. However, significant limitations affect current treatment options and clinical demand for bone grafts continues to rise due to conditions such as trauma, cancer, infection and arthritis. Developing bioactive three-dimensional (3D) scaffolds to support bone regeneration has therefore become a key area of focus within bone tissue engineering (BTE). A variety of materials and manufacturing methods including 3D printing have been used to create novel alternatives to traditional bone grafts. However, individual groups of materials including polymers, ceramics and hydrogels have been unable to fully replicate the properties of bone when used alone. Favourable material properties can be combined and bioactivity improved when groups of materials are used together in composite 3D scaffolds. This review will therefore consider the ideal properties of bioactive composite 3D scaffolds and examine recent use of polymers, hydrogels, metals, ceramics and bio-glasses in BTE. Scaffold fabrication methodology, mechanical performance, biocompatibility, bioactivity, and potential clinical translations will be discussed.

Tremendous progress in stem cell biology has resulted in a major current focus on effective modalities to promote directed cellular behavior for clinical therapy. The fundamental principles of tissue engineering are aimed at providing soluble and insoluble biological cues to promote these directed biological responses. Better understanding of extracellular matrix functions is ensuring optimal adhesive substrates to promote cell mobility and a suitable physical niche to direct stem cell responses. Further, appreciation of the roles of matrix constituents as morphogen cues, termed matrikines or matricryptins, are also now being directly exploited in biomaterial design. These insoluble topological cues can be presented at both micro- and nanoscales with specific fabrication techniques. Progress in development and molecular biology has described key roles for a range of biological molecules, such as proteins, lipids, and nucleic acids, to serve as morphogens promoting directed behavior in stem cells. Controlled-release systems involving encapsulation of bioactive agents within polymeric carriers are enabling utilization of soluble cues. Using our efforts at dental craniofacial tissue engineering, this narrative review focuses on outlining specific biomaterial fabrication techniques, such as electrospinning, gas foaming, and 3D printing used in combination with polymeric nano- or microspheres. These avenues are providing unprecedented therapeutic opportunities for precision bioengineering for regenerative applications.

Articular cartilage has a very limited ability to self-heal after injury or degeneration due to its low cellularity, poor proliferative activity, and avascular nature. Current clinical options are able to alleviate patient suffering, but cannot sufficiently regenerate the lost tissue. Biomimetic scaffolds that recapitulate the important features of the extracellular matrix (ECM) of cartilage are hypothesized to be advantageous in supporting cell growth, chondrogenic differentiation, and integration of regenerated cartilage with native cartilage, ultimately restoring the injured tissue to its normal function. It remains a challenge to support and maintain articular cartilage regenerated by bone marrow-derived mesenchymal stem cells (BMSCs), which are prone to hypertrophy and endochondral ossification after implantation in vivo. In the present work, a nanofibrous poly(l-lactic acid) (NF PLLA) scaffold developed by our group was utilized because of the desired highly porous structure, high interconnectivity, and collagen-like NF architecture to support rabbit BMSCs for articular cartilage regeneration. We further hypothesized that matrilin-3 (MATN3), a non-collagenous, cartilage-specific ECM protein, would enhance the microenvironment of the NF PLLA scaffold for cartilage regeneration and maintain the cartilage property. To test this hypothesis, we seeded BMSCs on the NF PLLA scaffold with or without MATN3. We found that MATN3 suppresses hypertrophy in this 3D culture system in vitro. Subcutaneous implantation of the chondrogenic cell/scaffold constructs in a nude mouse model showed that pretreatment with MATN3 was able to maintain chondrogenesis and prevent hypertrophy and endochondral ossification in vivo. These results demonstrate that the porous NF PLLA scaffold treated with MATN3 represents an advantageous 3D microenvironment for cartilage regeneration and phenotype maintenance, and is a promising strategy for articular cartilage repair.

Articular cartilage defects, caused by trauma, inflammation, or joint instability, may ultimately lead to debilitating pain and disability. Bone marrow-derived mesenchymal stem cells (BMSCs) are an attractive cell source for articular cartilage tissue engineering. However, chondrogenic induction of BMSCs is often accompanied by undesired hypertrophy, which can lead to calcification and ultimately damage the cartilage. Therefore, a therapy to prevent hypertrophy and endochondral ossification is of paramount importance to adequately regenerate articular cartilage. We hypothesized that MATN3 (a non-collagenous ECM protein expressed exclusively in cartilage) may improve regeneration of articular cartilage with BMSCs by maintaining chondrogenesis and preventing hypertrophic transition in an ECM mimicking nanofibrous scaffold. Our results showed that the administration of MATN3 to the cell/nanofibrous scaffold constructs favorably maintained chondrogenesis and prevented hypertrophy/endochondral ossification in the chondrogenic constructs in vitro and in vivo. The combination of nanofibrous PLLA scaffolds and MATN3 treatment provides a very promising strategy to generate chondrogenic grafts with phenotypic stability for articular cartilage repair.

This Article reports the fabrication and characterization of composite micro-nanostructured spiral scaffolds functionalized with nanofibers and hydroxyapatite (HA) for bone regeneration. The spiral poly(lactic acid-co-glycolic acid) (PLGA) porous microstructure was coated with sparsely spaced PLGA nanofibers and HA to enhance surface area and bioactivity. Polyelectrolyte-based HA coating in a layer-by-layer (LBL) fashion allowed 10-70 μM Ca²⁺/mm² incorporation. These scaffolds provided a controlled release of Ca²⁺ ions up to 60 days with varied release kinetics accounting up to 10-50 μg. Spiral scaffolds supported superior adhesion, proliferation, and osteogenic differentiation of rat bone marrow stromal cells (MSCs) as compared to controls microstructures. Spiral micro-nanostructures supported homogeneous tissue ingrowth and resulted in bone-island formation in the center of the scaffold as early as 3 weeks in a rabbit ulnar bone defect model. In contrast, control cylindrical scaffolds showed tissue ingrowth only at the surface because of limitations in scaffold transport features.

In this study, we investigated the anti-inflammatory, odontogenic and pro-angiogenic effects of integrating simvastatin and nanofibrous poly(l-lactic acid) (NF-PLLA) scaffolds on dental pulp cells (DPCs). Highly porous NF-PLLA scaffolds that mimic the nanofibrous architecture of extracellular matrix were first fabricated, then seeded with human DPCs and cultured with 0.1 μM simvastatin and/or 10 μg/mL pro-inflammatory stimulator lipopolysaccharide (LPS). The gene expression of pro-inflammatory mediators (TNF-α, IL-1β and MMP-9 mRNA) and odontoblastic markers (ALP activity, calcium content, DSPP, DMP-1 and BMP-2 mRNA) were quantified after long-term culture in vitro. In addition, we evaluated the scaffold's pro-angiogenic potential after 24 h of in vitro co-culture with endothelial cells. Finally, we assessed the combined effects of simvastatin and NF-PLLA scaffolds in vivo using a subcutaneous implantation mouse model. The in vitro studies demonstrated that, compared with the DPC/NF-PLLA scaffold constructs cultured only with pro-inflammatory stimulator LPS, adding simvastatin significantly repress the expression of pro-inflammatory mediators. Treating LPS+ DPC/NF-PLLA constructs with simvastatin also reverted the negative effects of LPS on expression of odontoblastic markers in vitro and in vivo. Western blot analysis demonstrated that these effects were related to a reduction in NFkBp65 phosphorylation and up-regulation of PPARγ expression, as well as to increased phosphorylation of pERK1/2 and pSmad1, mediated by simvastatin on LPS-stimulated DPCs. The DPC/NF-PLLA constructs treated with LPS/simvastatin also led to an increase in vessel-like structures, correlated with increased VEGF expression in both DPSCs and endothelial cells. Therefore, the combination of low dosage simvastatin and NF-PLLA scaffolds appears to be a promising strategy for dentin regeneration with inflamed dental pulp tissue, by minimizing the inflammatory reaction and increasing the regenerative potential of resident stem cells.

The regeneration potential of stem cells is dependent on their microenvironment. In this study, we investigated the effect of the microenvironment of dental pulp stem cells (DPSCs), including 3D structure of a macroporous and nanofibrous scaffold, the inflammatory stimulus lipopolysaccharide (LPS) and a biological molecule simvastatin, on their regenerative potential of mineralized dentin tissue. The results demonstrated that LPS upregulated inflammatory mediators and suppressed the odontogenic potential of DPSCs. Known as a lipid-lowing agent, simvastatin was excitingly found to repress the expression of pro-inflammatory mediators, up-regulate odontoblastic markers, and exert a pro-angiogenic effect on endothelial cells, resulting in enhanced vascularization and mineralized dentin tissue regeneration in a biomimetic 3D tissue engineering scaffold. This novel finding is significant for the fields of stem cells, inflammation and dental tissue regeneration.

Native tissue is naturally comprised of highly-ordered cell-matrix assemblies in a multi-hierarchical way, and the nano/submicron alignment of fibrous matrix is found to be significant in supporting cellular functionalization. In this study, a self-designed wet-spinning device appended with a rotary receiving pool was used to continuously produce shear-patterned hydrogel microfibers with aligned submicron topography. The process that the flow-induced shear force reshapes the surface of hydrogel fiber into aligned submicron topography was systematically analysed. Afterwards, the effect of fiber topography on cellular longitudinal spread and elongation was investigated by culturing rat neuron-like PC12 cells and human osteosarcoma MG63 cells with the spun hydrogel microfibers, respectively. The results suggested that the stronger shear flow force would lead to more distinct aligned submicron topography on fiber surface, which could induce cell orientation along with fiber axis and therefore form the cell-matrix dual-alignment. Finally, a multi-hierarchical tissue-like structure constructed by dual-oriented cell-matrix assemblies was fabricated based on this wet-spinning method. This work is believed to be a potentially novel biofabrication scheme for bottom-up constructing of engineered linear tissue, such as nerve bundle, cortical bone, muscle and hepatic cord.

The aim of this study is to investigate the behaviour of iPSc derived from dental stem cells in terms of initial adhesion, differentiation potential on differently surface-treated titanium disc.

iPSc derived from human gingival fibroblasts (hGFs) were established using 4-reprogramming factors transduction with Sendai virus. The hGF-iPSc established in this study exhibited the morphology and growth properties similar to human embryonic stem (ES) cells and expressed pluripotency makers. Alkaline Phosphatase (AP) staining, Embryoid Body (EB) formation and in vitro differentiation and karyotyping further confirmed pluripotency of hGF-iPSc. Then, hGF-iPSc were cultured on machined- and Sandblasted and acid etched (SLA)-treated titanium discs with osteogenic induction medium and their morphological as well as quantitative changes according to different surface types were investigated using Alizrin Red S staining, Scanning electron microscopy (SEM), Flow cytometry and RT-PCR.

Time-dependent and surface-dependent morphological changes as well as quantitative change in osteogenic differentiation of hGF-iPSc were identified and osteogenic gene expression of hGF-iPSc cultured on SLA-treated titanium disc found to be greater than machined titanium disc, suggesting the fate of hGF-iPSc may be determined by the characteristics of surface to which hGF-iPSc first adhere.

iPSc derived from dental stem cell can be one of the most promising and practical cell sources for personalized regenerative dentistry and their morphological change as well as quantitative change in osteogenic differentiation according to different surface types may be further utilized for future clinical application incorporated with dental implant.

Bone tissue engineering combines biomaterials with biologically active factors and cells to hold promise for reconstructing craniofacial defects. In this study the biological activity of biphasic hydroxyapatite ceramics (HA; a bone substitute that is a mixture of hydroxyapatite and β-tricalcium phosphate in fixed ratios) was characterized (1) in vitro by assessing the growth of MC3T3 mouse osteoblast lineage cells, (2) in ovo by using the chick chorioallantoic membrane (CAM) assay and (3) in an in vivo pig animal model. Biocompatibility, bioactivity, bone formation and biomaterial degradation were detected microscopically and by radiology and histology. HA ceramics alone demonstrated great biocompatibility on the CAM as well as bioactivity by increased proliferation and alkaline phosphatase secretion of mouse osteoblasts. The in vivo implantation of HA ceramics with bone marrow mesenchymal stem cells (MMSCs) showed de novo intramembranous bone healing of critical-size bone defects in the right lateral side of pig mandibular bodies after 3 and 9 weeks post-implantation. Compared with the HA ceramics without MMSCs, the progress of bone formation was slower with less-developed features. This article highlights the clinical use of microporous biphasic HA ceramics despite the unusually shaped elongated micropores with a high length/width aspect ratio (up to 20) and absence of preferable macropores (>100 µm) in bone regenerative medicine.

Biodegradable polymer microspheres have emerged as cell carriers for the regeneration and repair of irregularly shaped tissue defects due to their injectability, controllable biodegradability and capacity for drug incorporation and release. Notably, recent advances in nanotechnology allowed the manipulation of the physical and chemical properties of the microspheres at the nanoscale, creating nanostructured microspheres mimicking the composition and/or structure of natural extracellular matrix. These nanostructured microspheres, including nanocomposite microspheres and nanofibrous microspheres, have been employed as cell carriers for tissue regeneration. They enhance cell attachment and proliferation, promote positive cell-carrier interactions and facilitate stem cell differentiation for target tissue regeneration. This review highlights the recent advances in nanostructured microspheres that are employed as injectable, biomimetic and cell-instructive cell carriers.

Scaffolds for tissue engineering (TE) which closely mimic the physicochemical properties of the natural extracellular matrix (ECM) have been proven to advantageously favor cell attachment, proliferation, migration and new tissue formation. Recently, as a valuable alternative, a bottom-up TE approach utilizing cell-loaded micrometer-scale modular components as building blocks to reconstruct a new tissue in vitro or in vivo has been proved to demonstrate a number of desirable advantages compared with the traditional bulk scaffold based top-down TE approach. Nevertheless, micro-components with an ECM-mimicking nanofibrous structure are still very scarce and highly desirable. Chitosan (CS), an accessible natural polymer, has demonstrated appealing intrinsic properties and promising application potential for TE, especially the cartilage tissue regeneration. According to this background, we report here the fabrication of chitosan microspheres with an ECM-mimicking nanofibrous structure for the first time based on a physical gelation process. By combining this physical fabrication procedure with microfluidic technology, uniform CS microspheres (CMS) with controlled nanofibrous microstructure and tunable sizes can be facilely obtained. Especially, no potentially toxic or denaturizing chemical crosslinking agent was introduced into the products. Notably, in vitro chondrocyte culture tests revealed that enhanced cell attachment and proliferation were realized, and a macroscopic 3D geometrically shaped cartilage-like composite can be easily constructed with the nanofibrous CMS (NCMS) and chondrocytes, which demonstrate significant application potential of NCMS as the bottom-up cell-carrier components for cartilage tissue engineering.

Stem cell differentiation is regulated by multiple signaling events. Recent technical advances have revealed that differentiated cells can be reprogrammed into stem cells. The signals involved in stem cell programming are of major interest in stem cell research. The signaling mechanisms involved in regulating stem cell reprogramming and differentiation are the subject of intense study in the field of life sciences. In this review, the molecular interactions and signaling pathways related to stem cell differentiation are discussed.

Recent progress in nanotechnology has stimulated the development of multifunctional biomaterials for tissue engineering applications. Synergistic interactions between nanomaterials and stem cell engineering offer numerous possibilities to address some of the daunting challenges in regenerative medicine, such as controlling trigger differentiation, immune reactions, limited supply of stem cells, and engineering complex tissue structures. Specifically, the interactions between stem cells and their microenvironment play key roles in controlling stem cell fate, which underlines therapeutic success. However, the interactions between nanomaterials and stem cells are not well understood, and the effects of the nanomaterials shape, surface morphology, and chemical functionality on cellular processes need critical evaluation. In this Review, focus is put on recent development in nanomaterial-stem cell interactions, with specific emphasis on their application in regenerative medicine. Further, the emerging technologies based on nanomaterials developed over the past decade for stem cell engineering are reviewed, as well as the potential applications of these nanomaterials in tissue regeneration, stem cell isolation, and drug/gene delivery. It is anticipated that the enhanced understanding of nanomaterial-stem cell interactions will facilitate improved biomaterial design for a range of biomedical and biotechnological applications.

Human pluripotent stem cells (hPSCs) are a promising cell source with pluripotency and capacity to differentiate into all human somatic cell types. Designing simple and safe biomaterials with an innate ability to induce osteoblastic lineage from hPSCs is desirable to realize their clinical adoption in bone regenerative medicine. To address the issue, here we developed a fully defined synthetic peptides-decorated two-dimensional (2D) microenvironment via polydopamine (pDA) chemistry and subsequent carboxymethyl chitosan (CMC) grafting to enhance the culture and osteogenic potential of hPSCs in vitro. The hPSCs including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) were successfully cultured on the peptides-decorated surface without Matrigel and ECM protein coating and underwent promoted osteogenic differentiation in vitro, determined from the alkaline phosphate (ALP) activity, gene expression, and protein production as well as calcium deposit amount. It was found that directed osteogenic differentiation of hPSCs was achieved through a peptides-decorated niche. This chemically defined and safe 2D microenvironment, which facilitates proliferation and osteo-differentiation of hPSCs, not only helps to accelerate the translational perspectives of hPSCs but also provides tissue-specific functions such as directing stem cell differentiation commitment, having great potential in bone tissue engineering and opening new avenues for bone regenerative medicine.

Human embryonic stem cells (hESCs) are an exciting cell source as they offer an unlimited supply of cells that can differentiate into all cell types for regenerative medicine applications. To date, there has been no report on hESCs with calcium phosphate cement (CPC) scaffolds for bone regeneration in vivo. The objectives of this study were to: (i) investigate hESCs for bone regeneration in vivo in critical-sized cranial defects in rats; and (ii) determine the effects of cell seeding and platelets in macroporous CPC on new bone and blood vessel formation. hESCs were cultured to yield mesenchymal stem cells (MSCs), which underwent osteogenic differentiation. Four groups were tested in rats: (i) CPC control without cells; (ii) CPC with hESC-derived MSCs (CPC+hESC-MSC); (iii) CPC with hESC-MSCs and 30% human platelet concentrate (hPC) (CPC+hESC-MSC+30% hPC); and (iv) CPC+hESC-MSC+50% hPC. In vitro, MSCs were derived from embryoid bodies of hESCs. Cells on CPC were differentiated into the osteogenic lineage, with highly elevated alkaline phosphatase and osteocalcin expressions, as well as mineralization. At 12weeks in vivo, the groups with hESC-MSCs and hPC had three times as much new bone as, and twice the blood vessel density of, the CPC control. The new bone in the defects contained osteocytes and blood vessels, and the new bone front was lined with osteoblasts. The group with 30% hPC and hESC-MSCs had a blood vessel density that was 49% greater than the hESC-MSC group without hPC, likely due to the various growth factors in the platelets enhancing both new bone and blood vessel formation. In conclusion, hESCs are promising for bone tissue engineering, and hPC can enhance new bone and blood vessel formation. Macroporous CPC with hESC-MSCs and hPC may be useful for bone regeneration in craniofacial and orthopedic applications.

The physical and chemical properties of a matrix play an important role in determining various cellular behaviors, including lineage specificity. We demonstrate that the differentiation commitment of human embryonic stem cells (hESCs), both in vitro and in vivo, can be solely achieved through synthetic biomaterials. hESCs were cultured using mineralized synthetic matrices mimicking a calcium phosphate (CaP)-rich bone environment differentiated into osteoblasts in the absence of any osteogenic inducing supplements. When implanted in vivo, these hESC-laden mineralized matrices contributed to ectopic bone tissue formation. In contrast, cells within the corresponding non-mineralized matrices underwent either osteogenic or adipogenic fate depending upon the local cues present in the microenvironment. To our knowledge, this is the first demonstration where synthetic matrices are shown to induce terminal cell fate specification of hESCs exclusively by biomaterial-based cues both in vitro and in vivo. Technologies that utilize tissue specific cell-matrix interactions to control stem cell fate could be a powerful tool in regenerative medicine. Such approaches can be used as a tool to advance our basic understanding and assess the translational potential of stem cells.

Calcium phosphate cements (CPCs) have excellent biocompatibility and osteoconductivity for dental, craniofacial, and orthopedic applications. This article reviews recent developments in stem cell delivery via CPC for bone regeneration. This includes: (1) biofunctionalization of the CPC scaffold, (2) co-culturing of osteoblasts/endothelial cells and prevascularization of CPC, (3) seeding of CPC with different stem cell species, (4) human umbilical cord mesenchymal stem cell (hUCMSC) and bone marrow MSC (hBMSC) seeding on CPC for bone regeneration, and (5) human embryonic stem cell (hESC) and induced pluripotent stem cell (hiPSC) seeding with CPC for bone regeneration. Cells exhibited good attachment/proliferation in CPC scaffolds. Stem-cell-CPC constructs generated more new bone and blood vessels in vivo than did the CPC control without cells. hUCMSCs, hESC-MSCs, and hiPSC-MSCs in CPC generated new bone and blood vessels similar to those of hBMSCs; hence, they were viable cell sources for bone engineering. CPC with hESC-MSCs and hiPSC-MSCs generated new bone two- to three-fold that of the CPC control. Therefore, this article demonstrates that: (1) CPC scaffolds are suitable for delivering cells; (2) hUCMSCs, hESCs, and hiPSCs are promising alternatives to hBMSCs, which require invasive procedures to harvest with limited cell quantity; and (3) stem-cell-CPC constructs are highly promising for bone regeneration in dental, craniofacial, and orthopedic applications.