Stepwise dual-release microparticles of BMP-4 and SCF in induced pluripotent stem cell spheroids enhance differentiation into hematopoietic stem cells.

Recently, the formation of three-dimensional (3D) cell aggregates known as embryoid bodies (EBs) grown in media supplemented with HSC-specific morphogens has been utilized for the directed differentiation of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), into clinically relevant hematopoietic stem cells (HSCs). However, delivering growth factors and nutrients have become ineffective in inducing synchronous differentiation of cells due to their 3D conformation. Moreover, irregularly sized EBs often lead to the formation of necrotic cores in larger EBs, impairing differentiation. Here, we developed two gelatin microparticles (GelMPs) with different release patterns and two HSC-related growth factors conjugated to them. Slow and fast releasing GelMPs were conjugated with bone morphogenic factor-4 (BMP-4) and stem cell factor (SCF), respectively. The sequential presentation of BMP-4 and SCF in GelMPs resulted in efficient and effective hematopoietic differentiation, shown by the enhanced gene and protein expression of several mesoderm and HSC-related markers, and the increased concentration of released HSC-related cytokines. In the present study, we were able to generate CD34+, CD133+, and FLT3+ cells with similar cellular and molecular morphology as the naïve HSCs that can produce colony units of different blood cells, in vitro.

Therapeutic potential of mesenchymal stem cells from human iPSC-derived teratomas for osteochondral defect regeneration.

Human induced pluripotent stem cells (iPSCs) hold great promise for personalized medicine, as they can be differentiated into specific cell types, especially mesenchymal stem cells (MSCs). Therefore, our study sought to assess the feasibility of deriving MSCs from teratomas generated from human iPSCs. Teratomas serve as a model to mimic multilineage human development, thus enriching specific somatic progenitors and stem cells. Here, we discovered a small, condensed mass of MSCs within iPSC-generated teratomas. Afterward, we successfully isolated MSCs from this condensed mass, which was a byproduct of teratoma development. To evaluate the characteristics and cell behaviors of iPSC-derived MSCs (iPSC-MSCs), we conducted comprehensive assessments using qPCR, immunophenotype analysis, and cell proliferation-related assays. Remarkably, iPSC-MSCs exhibited an immunophenotype resembling that of conventional MSCs, and they displayed robust proliferative capabilities, similar to those of higher pluripotent stem cell-derived MSCs. Furthermore, iPSC-MSCs demonstrated the ability to differentiate into multiple lineages in vitro. Finally, we evaluated the therapeutic potential of iPSC-MSCs using an osteochondral defect model. Our findings demonstrated that teratomas are a promising source for the isolation of condensed MSCs. More importantly, our results suggest that iPSC-MSCs derived from teratomas possess the capacity for tissue regeneration, highlighting their promise for future therapeutic applications.

Emerging nano-scale delivery systems for the treatment of osteoporosis.

Osteoporosis is a pathological condition characterized by an accelerated bone resorption rate, resulting in decreased bone density and increased susceptibility to fractures, particularly among the elderly population. While conventional treatments for osteoporosis have shown efficacy, they are associated with certain limitations, including limited drug bioavailability, non-specific administration, and the occurrence of adverse effects. In recent years, nanoparticle-based drug delivery systems have emerged as a promising approach for managing osteoporosis. Nanoparticles possess unique physicochemical properties, such as a small size, large surface area-to-volume ratio, and tunable surface characteristics, which enable them to overcome the limitations of conventional therapies. These nanoparticles offer several advantages, including enhanced drug stability, controlled release kinetics, targeted bone tissue delivery, and improved drug bioavailability. This comprehensive review aims to provide insights into the recent advancements in nanoparticle-based therapy for osteoporosis. It elucidates the various types of nanoparticles employed in this context, including silica, polymeric, solid lipid, and metallic nanoparticles, along with their specific processing techniques and inherent properties that render them suitable as potential drug carriers for osteoporosis treatment. Furthermore, this review discusses the challenges and future suggestions associated with the development and translation of nanoparticle drug delivery systems for clinical use. These challenges encompass issues such as scalability, safety assessment, and regulatory considerations. However, despite these challenges, the utilization of nanoparticle-based drug delivery systems holds immense promise in revolutionizing the field of osteoporosis management by enabling more effective and targeted therapies, ultimately leading to improved patient outcomes.

Progress and emerging techniques for biomaterial-based derivation of mesenchymal stem cells (MSCs) from pluripotent stem cells (PSCs).

The use of mesenchymal stem cells (MSCs) for clinical purposes has skyrocketed in the past decade. Their multilineage differentiation potentials and immunomodulatory properties have facilitated the discovery of therapies for various illnesses. MSCs can be isolated from infant and adult tissue sources, which means they are easily available. However, this raises concerns because of the heterogeneity among the various MSC sources, which limits their effective use. Variabilities arise from donor- and tissue-specific differences, such as age, sex, and tissue source. Moreover, adult-sourced MSCs have limited proliferation potentials, which hinders their long-term therapeutic efficacy. These limitations of adult MSCs have prompted researchers to develop a new method for generating MSCs. Pluripotent stem cells (PSCs), such as embryonic stem cells and induced PSCs (iPSCs), can differentiate into various types of cells. Herein, a thorough review of the characteristics, functions, and clinical importance of MSCs is presented. The existing sources of MSCs, including adult- and infant-based sources, are compared. The most recent techniques for deriving MSCs from iPSCs, with a focus on biomaterial-assisted methods in both two- and three-dimensional culture systems, are listed and elaborated. Finally, several opportunities to develop improved methods for efficiently producing MSCs with the aim of advancing their various clinical applications are described.

Therapeutic potential of epiphyseal growth plate cells for bone regeneration in an osteoporosis model.

Bone growth occurs in the epiphyseal growth plate (EGP) and epiphyseal growth plate cells (EGPCs) exist in EGP. EGPCs, including skeletal stem cells (SSCs), are cells that induce bone growth and development through endochondral ossification. Recently, the superiority of bone regeneration through endochondral ossification has been reported. Our study compared EGPCs with bone marrow-derived mesenchymal stem cells (BM-MSCs) and suggested the therapeutic potential of new bone regeneration. In this study, we analyzed the characteristics between EGPCs and BM-MSCs based on morphological characteristics and molecular profiles. EGPCs expressed chondrogenic and osteogenic markers higher than BM-MSCs. Additionally, in co-culture with BM-MSCs, EGPCs induced an increase in chondrogenic, osteogenic, and hypertrophic markers of BM-MSCs. Finally, EGPCs induced higher bone regeneration than BM-MSCs in the osteoporosis model. Overall, we suggest the possibility of EGPCs as cell therapy for effective bone regeneration.

SPRY4 acts as an indicator of osteoarthritis severity and regulates chondrocyte hypertrophy and ECM protease expression.

Osteoarthritis (OA) causes serious changes in the metabolic and signaling pathways of chondrocytes, including the mitogen-activated protein kinase (MAPK) pathway. However, the role of sprouty RTK signaling antagonist 4 (SPRY4), an inhibitor of MAPK, in the human cartilage tissues and chondrocytes remains to be understood. Here, using SPRY4 gene delivery into healthy and degenerated chondrocytes, we elucidated the role of SPRY4 in preventing chondrocyte hypertrophy. In addition to using the human cartilage tissues with the destabilization of the medial meniscus (DMM) model in Sprague-Dawley (SD) rats, the role of SPRY4 in cartilage tissues and chondrocytes was explored through their molecular and histological analyses. In order to determine the effects of SPRY4 on healthy human chondrocyte hypertrophy, small interfering RNA (siRNA) was used to knock down SPRY4. Lentiviral transduction of SPRY4 into degenerated human chondrocytes allowed us to investigate its ability to prevent hypertrophy. SPRY4 expression levels were higher in healthy human cartilage tissue and chondrocytes than in degenerated human cartilage tissues and hypertrophy-induced chondrocytes. The knockdown of SPRY4 in healthy chondrocytes caused an increase in hypertrophy, senescence, reactive oxygen species (ROS) production, and extracellular matrix (ECM) protease expression. However, all these factors decreased upon overexpression of SPRY4 in degenerated chondrocytes via regulation of the MAPK signaling pathway. We conclude that SPRY4 is a crucial indicator of osteoarthritis (OA) severity and could play an important role in preventing OA in the cartilage by inhibiting chondrocyte hypertrophy.

Matrilin3/TGFß3 gelatin microparticles promote chondrogenesis, prevent hypertrophy, and induce paracrine release in MSC spheroid for disc regeneration.

Degenerative disc disease (DDD) is the leading cause of excruciating lower back pain and disability in adults worldwide. Among the current treatments for DDD, cell-based therapies such as the injection of both disc- and non-disc-derived chondrocytes have shown significant improvements in the patients' condition. However, further advancement of these therapies is required to not only ensure a supply of healthy chondrocytes but also to promote regeneration of the defective cells in the injury site. Here, we report that the incorporation of gelatin microparticles coloaded with transforming growth factor beta 3 and matrilin 3 promoted chondrogenic differentiation of adipose-derived mesenchymal stem cell spheroids while preventing hypertrophy and terminal differentiation of cells. Moreover, these composite spheroids induced the release of chondrogenic cytokines that, in turn, promoted regeneration of degenerative chondrocytes in vitro. Finally, injections of these composite spheroids in a rat model of intervertebral disc disease promoted restoration of the chondrogenic properties of the cells, thereby allowing regeneration of the chondrogenic tissue in vivo.

Functional Duality of Chondrocyte Hypertrophy and Biomedical Application Trends in Osteoarthritis.

Chondrocyte hypertrophy is one of the key indicators in the progression of osteoarthritis (OA). However, compared with other OA indications, such as cartilage collapse, sclerosis, inflammation, and protease activation, the mechanisms by which chondrocyte hypertrophy contributes to OA remain elusive. As the pathological processes in the OA cartilage microenvironment, such as the alterations in the extracellular matrix, are initiated and dictated by the physiological state of the chondrocytes, in-depth knowledge of chondrocyte hypertrophy is necessary to enhance our understanding of the disease pathology and develop therapeutic agents. Chondrocyte hypertrophy is a factor that induces OA progression; it is also a crucial factor in the endochondral ossification. This review elaborates on this dual functionality of chondrocyte hypertrophy in OA progression and endochondral ossification through a description of the characteristics of various genes and signaling, their mechanism, and their distinguishable physiological effects. Chondrocyte hypertrophy in OA progression leads to a decrease in chondrogenic genes and destruction of cartilage tissue. However, in endochondral ossification, it represents an intermediate stage at the process of differentiation of chondrocytes into osteogenic cells. In addition, this review describes the current therapeutic strategies and their mechanisms, involving genes, proteins, cytokines, small molecules, three-dimensional environments, or exosomes, against the OA induced by chondrocyte hypertrophy. Finally, this review proposes that the contrasting roles of chondrocyte hypertrophy are essential for both OA progression and endochondral ossification, and that this cellular process may be targeted to develop OA therapeutics.

Efficient Isolation and Enrichment of Mesenchymal Stem Cells from Human Embryonic Stem Cells by Utilizing the Interaction between Integrin α5ß1 and Fibronectin.

Human pluripotent stem cells (hPSCs) are a potent source of clinically relevant mesenchymal stem cells (MSCs) that confer functional and structural benefits in cell therapy and tissue regeneration. Obtaining sufficient numbers of MSCs in a short period of time and enhancing the differentiation potential of MSCs can be offered the potential to improve the regenerative activity of MSCs therapy. In addition, the underlying processes in the isolation and derivation of MSCs from hPSCs are still poorly understood and controlled. To overcome these clinical needs, an efficient and simplified technique on the isolation of MSCs from spontaneously differentiated human embryonic stem cells (hESCs) via integrin α5ß1 (fibronectin (FN) receptor)-to-FN interactions (hESC-FN-MSCs) is successfully developed. It is demonstrated that hESC-FN-MSCs exhibit a typical MSC surface phenotype, cellular morphology, with the whole transcriptome similar to conventional adult MSCs; but show higher proliferative capacity, more efficient trilineage differentiation, enhanced cytokine secretion, and attenuated cellular senescence. In addition, the therapeutic potential and regenerative capacity of the isolated hESC-FN-MSCs are confirmed by in vitro and in vivo multilineage differentiation. This novel method will be useful in the generation of abundant amounts of clinically relevant MSCs for stem cell therapeutics and regenerative medicine.

Engineering and Functionalization of Gelatin Biomaterials: From Cell Culture to Medical Applications.

Health care and medicine were revolutionized in recent years by the development of biomaterials, such as stents, implants, personalized drug delivery systems, engineered grafts, cell sheets, and other transplantable materials. These materials not only support the growth of cells before transplantation but also serve as replacements for damaged tissues in vivo. Among the various biomaterials available, those made from natural biological sources such as extracellular proteins (collagen, fibronectin, laminin) have shown significant benefits, and thus are widely used. However, routine biomaterial-based research requires copious quantities of proteins and the use of pure and intact extracellular proteins could be highly cost ineffective. Gelatin is a molecular derivative of collagen obtained through the irreversible denaturation of collagen proteins. Gelatin shares a very close molecular structure and function with collagen and thus is often used in cell and tissue culture to replace collagen for biomaterial purposes. Recent technological advancements such as additive manufacturing, rapid prototyping, and three-dimensional printing, in general, have resulted in great strides toward the generation of functional gelatin-based materials for medical purposes. In this review, the structural and molecular similarities of gelatin to other extracellular matrix proteins are compared and analyzed. Current strategies for gelatin crosslinking and production are described and recent applications of gelatin-based biomaterials in cell culture and tissue regeneration are discussed. Finally, recent improvements in gelatin-based biomaterials for medical applications and future directions are elaborated. Impact statement In this study, we described gelatin's biochemical properties and compared its advantages and drawbacks over other extracellular matrix proteins and polymers used for biomaterial application. We also described how gelatin can be used with other polymers in creating gelatin composite materials that have enhanced mechanical properties, increased biocompatibility, and boosted bioactivity, maximizing its benefits for biomedical purposes. The article is relevant, as it discussed not only the chemistry of gelatin, but also listed the current techniques in gelatin/biomaterial manufacturing and described the most recent trends in gelatin-based biomaterials for biomedical applications.

Suppression of SPRY4 Promotes Osteogenic Differentiation and Bone Formation of Mesenchymal Stem Cell.

The directed differentiation of human adipose-derived stem cells (hASCs) into different cell types has shown great therapeutic potential in treating various diseases. To maximize the therapeutic potentials, researchers have tried manipulating master transcriptional genes that promote efficient differentiation of mesenchymal stem cells (MSCs) such as the MAPK/ERK signaling pathway. Sprouty (SPRY) is a family of proteins that are known to inhibit the MAPK/ERK signaling pathway. Although the role of some SPRY isoforms in MSC differentiation is known, the function of SPRY4 isoform has not been fully elucidated. In the present study, the role of SPRY4 in the multilineage differentiation of hASCs has been elucidated. To investigate the role of SPRY4 in hASC differentiation and tissue regeneration, we performed a transient knockdown of SPRY expression via a small interfering RNA (siSPRY4). Western blot and quantitative polymerase chain reaction results revealed that the treatment of siSPRY4 before induction of differentiation had no significant effect on adipogenic, but reduced chondrogenic, differentiation of hASCs. Interestingly, SPRY4 transient knockdown had a significant effect on the osteogenic differentiation as indicated by the increased messenger RNA (mRNA) and protein expression of osteogenic markers such as alkaline phosphatase (ALP; 2.3-fold) and osteopontin (OPN; 3.5-fold) and increased calcium deposition measured via Alizarin red staining (3.3-fold). Moreover, in vivo tissue regeneration of siSPRY4-treated hASCs in ectopic bone formation and calvarial defect mouse models showed higher bone volume (5.24-fold) and trabecular number (4.59-fold) assessed via histological and microcomputed tomography analyses. We also determined that the enhanced osteogenic differentiation in SPRY4-treated hASCs was due to the induction of ERK1/2 phosphorylation. Taken together, our results suggest that the regulation of SPRY4 through MAPK signaling is a potentially critical aspect on the osteogenic differentiation of hASCs and for bone tissue regeneration, and thus, may be utilized as a potent technique in the development of effective bone therapeutics. Impact Statement This study tried to expand our current understanding of the osteogenic differentiation of mesenchymal stem cells. The transient downregulation of the SPRY4 expression via small interfering RNA (siRNA) showed significant enhancement of the osteogenic differentiation of adipose-derived stem cells via the induction of ERK 1/2 phosphorylation. This suggests the possible mechanism to maximize the potential of stem cell as therapeutics and has a great potential in treating various bone-related diseases.

Corrigendum: Oral Administration of α-Asarone Promotes Functional Recovery in Rats With Spinal Cord Injury.

[This corrects the article DOI: 10.3389/fphar.2018.00445.].

Oral Administration of α-Asarone Promotes Functional Recovery in Rats With Spinal Cord Injury.

α-asarone, a bioactive compound found in Acorus plant species, has been shown to exhibit neuroprotective, anti-oxidative, anti-inflammatory, and cognitive-enhancing effects. However, the effects of α-asarone on spinal cord injury (SCI) have not yet been elucidated. The present study investigated the effects of α-asarone on the mRNA of pro-inflammatory cytokines, macrophage polarization toward an anti-inflammatory M2 phenotype, and angiogenesis in rats with compressive SCI. α-Asarone was orally administered (10 mg/kg) once per day for 14 days following moderate static compression SCI. Compared to controls, α-asarone treatment significantly improved locomotor score, prevented neuroinflammation, and facilitated angiogenesis in the spinal cord at 14 days after SCI. Furthermore, α-asarone significantly reduced the TNF-α, IL-1ß, IL-6, monocyte chemoattractant protein 1 (MCP-1), macrophage inflammatory protein 2 (MIP-2), and inducible nitric oxide synthase (iNOS) levels but increased the IL-4, IL-10, and arginase 1 levels at 24 h after SCI. At 7 and 14 days after SCI, immunohistochemistry showed reduced reactive gliosis and neuroinflammation and an increased expression of M2 macrophage markers and angiogenesis. The results suggest that the inhibition of pro-inflammatory cytokines, macrophage polarization toward an anti-inflammatory M2 phenotype, and angiogenesis by α-asarone may be some of the mechanisms underlying the α-asarone-mediated neuroprotective effects on an injured spinal cord.

Current approaches in biomaterial-based hematopoietic stem cell niches.

Hematopoietic stem cells (HSCs) are multipotent progenitor cells that can differentiate and replenish blood and immune cells. While there is a growing demand for autologous and allogeneic HSC transplantation owing to the increasing incidence of hereditary and hematologic diseases, the low population of HSCs in cord-blood and bone marrow (the main source of HSCs) hinders their medical applicability. Several cytokine and growth factor-based methods have been developed to expand the HSCs in vitro; however, the expansion rate is low, or the expanded cells fail to survive upon engraftment. This is at least in part because the overly simplistic polystyrene culture substrates fail to fully replicate the microenvironments or niches where these stem cells live. Bone marrow niches are multi-dimensional, complex systems that involve both biochemical (cells, growth factors, and cytokines) and physiochemical (stiffness, O2 concentration, and extracellular matrix presentation) factors that regulate the quiescence, proliferation, activation, and differentiation of the HSCs. Although several studies have been conducted on in vitro HSC expansion via 2D and 3D biomaterial-based platforms, additional work is required to engineer an effective biomaterial platform that mimics bone marrow niches. In this study, the factors that regulate the HSC in vivo were explained and their applications in the engineering of a bone marrow biomaterial-based platform were discussed. In addition, current approaches, challenges, and the future direction of a biomaterial-based culture and expansion of the HSC were examined. STATEMENT OF SIGNIFICANCE: Hematopoietic stem cells (HSC) are multipotent cells that can differentiate and replace the blood and immune cells of the body. However, in vivo, there is a low population of these cells, and thus their use in biotherapeutic and medical applications is limited (i.e., bone marrow transplantation). In this review, the biochemical factors (growth factors, cytokines, co-existing cells, ECM, gas concentrations, and differential gene expression) that may regulate the over-all fate of HSC, in vivo, were summarized and discussed. Moreover, different conventional and recent biomaterial platforms were reviewed, and their potential in generating a biomaterial-based, BM niche-mimicking platform for the efficient growth and expansion of clinically relevant HSCs in-vitro, was discussed.

Osteogenic Potential of Tauroursodeoxycholic Acid as an Alternative to rhBMP-2 in a Mouse Spinal Fusion Model.

The non-union rate after lumbar spinal fusion is potentially as high as 48%. To support efficient bone regeneration, recombinant human bone morphogenetic protein-2 (rhBMP-2) is commonly used as it is regarded as the most potent bone-inducing molecule. However, recently, there have been increasing concerns on the use of rhBMP-2 such as serious complications, including seroma and heterotopic ossification, and the low quality of bone at the center of fusion mass. Thus, many studies were conducted to find and to develop a potential alternative to rhBMP-2. In this study, we investigated the osteogenic potential of tauroursodeoxycholic acid (TUDCA) in the mouse fusion model and compared its effects with rhBMP-2. Twenty-four mice underwent bilateral posterolateral lumbar spinal fusion bone formation at L4-L5. Collagen sponge infused with saline, TUDCA, or rhBMP-2 was implanted at the fusion area. Two and 4 weeks postimplantation, bone formation and tissue regeneration were evaluated via micro-computed tomography and histological analysis. Compared with the TUDCA-treated group, the rhBMP-2 treatment produced a higher amount of bone fusion formation after 2 weeks but also showed higher resorption of the centralized bone after 4 weeks. Interestingly, the TUDCA-treated group developed higher trabecular thickness compared with rhBMP-2 after 4 weeks. Moreover, TUDCA treatment showed distinct angiogenic activity in human umbilical vein endothelial cells as confirmed by an in vitro tube formation assay. Our findings suggest that TUDCA is comparable to rhBMP-2 in supporting bone regeneration and spinal bone formation fusion by increasing trabecular thickness and promoting angiogenesis. Finally, our results indicate that TUDCA can be utilized as a potential alternative to rhBMP-2.