Redifferentiation of genetically modified dedifferentiated chondrocytes in a microcavitary hydrogel.

OBJECTIVES: We genetically modified dedifferentiated chondrocytes (DCs) using lentiviral vectors and adenoviral vectors encoding TGF-ß3 (referred to as transgenic groups below) and encapsulated these DCs in the microcavitary hydrogel and investigated the combinational effect on redifferentiation of the genetically manipulated DCs. RESULTS: The Cell Counting Kit-8 data indicated that both transgenic groups exhibited significantly higher cell viability in the first week but inferior cell viability in the subsequent timepoints compared with those of the control group. Real-time polymerase chain reaction and western blot analysis results demonstrated that both transgenic groups had a better effect on redifferentiation to some extent, as evidenced by higher expression levels of chondrogenic genes, suggesting the validity of combination with transgenic DCs and the microcavitary hydrogel on redifferentiation. Although transgenic DCs with adenoviral vectors presented a superior extent of redifferentiation, they also expressed greater levels of the hypertrophic gene type X collagen. It is still worth further exploring how to deliver TGF-ß3 more efficiently and optimizing the appropriate parameters, including concentration and duration. CONCLUSIONS: The results demonstrated the better redifferentiation effect of DCs with the combinational use of transgenic TGF-ß3 and a microcavitary alginate hydrogel and implied that DCs would be alternative seed cells for cartilage tissue engineering due to their easily achieved sufficient cell amounts through multiple passages and great potential to redifferentiate to produce cartilaginous extracellular matrix.

Progress in microsphere-based scaffolds in bone/cartilage tissue engineering.

Bone/cartilage repair and regeneration have been popular and difficult issues in medical research. Tissue engineering is rapidly evolving to provide new solutions to this problem, and the key point is to design the appropriate scaffold biomaterial. In recent years, microsphere-based scaffolds have been considered suitable scaffold materials for bone/cartilage injury repair because microporous structures can form more internal space for better cell proliferation and other cellular activities, and these composite scaffolds can provide physical/chemical signals for neotissue formation with higher efficiency. This paper reviews the research progress of microsphere-based scaffolds in bone/chondral tissue engineering, briefly introduces types of microspheres made from polymer, inorganic and composite materials, discusses the preparation methods of microspheres and the exploration of suitable microsphere pore size in bone and cartilage tissue engineering, and finally details the application of microsphere-based scaffolds in biomimetic scaffolds, cell proliferation and drug delivery systems.

Progress in Osteochondral Regeneration with Engineering Strategies.

Osteoarthritis, the main cause of disability worldwide, involves not only cartilage injury but also subchondral bone injury, which brings challenges to clinical repair. Tissue engineering strategies provide a promising solution to this degenerative disease. Articular cartilage connects to subchondral bone through the osteochondral interfacial tissue, which has a complex anatomical architecture, distinct cell distribution and unique biomechanical properties. Forming a continuous and stable osteochondral interface between cartilage tissue and subchondral bone is challenging. Thus, successful osteochondral regeneration with engineering strategies requires intricately coordinated interplay between cells, materials, biological factors, and physical/chemical factors. This review provides an overview of the anatomical composition, microstructure, and biomechanical properties of the osteochondral interface. Additionally, the latest research on the progress related to osteochondral regeneration is reviewed, especially discussing the fabrication of biomimetic scaffolds and the regulation of biological factors for osteochondral defects.

Prospects of cell chemotactic factors in bone and cartilage tissue engineering.

INTRODUCTION: Tissue engineering has brought hope for the repair of bone and cartilage injury. As potential therapeutic molecules for use in tissue engineering, chemokines promote the development of cell-free tissue engineering, avoiding dilemmas faced by cell-based tissue engineering. The main role of chemokines in tissue engineering is to recruit progenitor/stem cells to the site of damaged tissue in vivo and induce differentiation into the corresponding tissue, thus remodeling tissue function. In recent years, many studies have demonstrated the great potential of chemokines in the regeneration and repair of various tissues, such as heart, bone and cartilage tissue. AREAS COVERED: The classification, structure, and function of chemokines and the application of several common chemokines in diseases, especially in bone/cartilage tissue regeneration are discussed. EXPERT OPINION: Many studies have demonstrated that the combinatory use of cell chemotactic factors (CCFs) and growth factors can exert synergistic effects on chondrogenesis and osteogenesis. With further understanding of biomaterials and the development of powerful bio-fabrication techniques, intelligent biomaterials will be created to meet the requirements for controlled bioactive factor release and biomimetic architecture. Also, a better understanding of the biological cascade reactions and pathways of CCFs is beneficial to guide the design of innovative biomaterials.

Effect of passage number of genetically modified TGF-ß3 expressing primary chondrocytes on the chondrogenesis of ATDC5 cells in a 3D coculture system.

Due to the lack of blood vessels, nerves and lymphatics, articular cartilage is difficult to repair once damaged. Tissue engineering is considered to be a potential strategy for cartilage regeneration. Successful tissue engineering strategies depend on the effective combination of biomaterials, seed cells and biological factors. In our previous study, a genetically modified coculture system with chondrocytes and ATDC5 cells in an alginate hydrogel has exhibited a superior ability to enhance chondrogenesis. In this study, we further evaluated the influence of chondrocytes at various passages on chondrogenesis in the coculture system. The results demonstrated that transfection efficiency was hardly influenced by the passage number of chondrocytes. The coculture system with passage 5 (P5) chondrocytes had a better effect on chondrogenesis of ATDC5 cells, while chondrocytes in this coculture system presented higher levels of dedifferentiation than other groups with P1 or P3 chondrocytes. Therefore, P5 chondrocytes were shown to be more suitable for the coculture system, as they accumulated in sufficient cell numbers with more passages and had a higher level of dedifferentiation, which was prone to form a favorable niche for chondrogenesis of ATDC5 cells. This study may provide fresh insights for future cartilage tissue engineering strategies with a combination of a coculture system and advanced biomaterials.

Progress of biomaterials for bone tumor therapy.

Bone tumors are currently a major clinical challenge. In recent decades, strategies using well-designed versatile biomaterials for the treatment of bone tumors have emerged and attracted extensive research interest. Suitable biomaterials not only facilitate repair for bone defects aroused by surgical intervention but also help deliver antineoplastic drugs to the target site or provide photothermal/magnetothermal therapy to kill bone tumor cells. Thus, the development of biomaterials exhibits a great perspective for future bone tumor treatment.We summarize the recent progress of versatile biomaterials for bone tumor therapy, with an emphasis on photothermal/magnetothermal therapy and drug delivery.With the further understanding and development of biomaterials, multifunctional biomaterials have been proposed for bone tumor treatment. Through the interdisciplinary cooperation from the fields of biomedicine, clinical medicine and engineering, multifunctional biomaterials will perfectly match individual bone defects in the clinic with low cost in the future.

The Role of Mechanical Regulation in Cartilage Tissue Engineering.

Due to the lack of vascular distribution and the slow metabolism, cartilage tissue cannot repair itself, which remains a huge challenge in cartilage regeneration. Tissue engineering using stem cells appears to be a promising method for cartilage repair. Tissue engineers demonstrated that mechanical stimulation can enhance the quality of engineered cartilage, making it more similar to natural cartilage in structure and function. In this review, we summarize recent studies on the role of mechanical stimuli in chondrogenesis, focusing on the applications of extrinsic mechanical loading and the studies on mechanical properties of biomaterials in cartilage tissue engineering. This review will provide fresh insights into the potential use of mechanical stimuli for clinical use.

Gene-modified BMSCs encapsulated with carboxymethyl cellulose facilitate osteogenesis in vitro and in vivo.

Critical size bone defects are one of the most serious complications in orthopedics due to the lack of effective osteogenesis treatment. We fabricated carboxymethyl cellulose with phenol moieties (CMC-ph) microcapsules loaded with gene-modified rat bone mesenchymal stem cells (rBMSCs) that secrete hBMP2 following doxycycline (DOX) induction. The results showed that the morphology of microcapsules was spherical, and their diameters have equally distributed in the range of 100-150 µm; the viability of rBMSCs was unchanged over time. Through real-time PCR and Western blot analyses, the rBMSCs in microcapsules were found to secrete hBMP2 and to have upregulated mRNA and protein expression of osteogenesis-related genes in vitro and in vivo. Furthermore, the in vivo results suggested that the group with the middle concentration of cells expressed the highest amount of osteogenic protein over time. In this study, we showed that gene-modified rBMSCs in CMC-ph microcapsules had good morphology and viability. The BMP2-BMSCs/CMC-Ph microcapsule system could upregulate osteogenic mRNA and protein in vitro and in vivo. Further analysis demonstrated that the medium concentration of cells had a suitable density for transplantation in nude mice. Therefore, BMP2-BMSCs/CMC-Ph microcapsule constructs have potential for bone regeneration in vivo.

Dedifferentiation: inspiration for devising engineering strategies for regenerative medicine.

Cell dedifferentiation is the process by which cells grow reversely from a partially or terminally differentiated stage to a less differentiated stage within their own lineage. This extraordinary phenomenon, observed in many physiological processes, inspires the possibility of developing new therapeutic approaches to regenerate damaged tissue and organs. Meanwhile, studies also indicate that dedifferentiation can cause pathological changes. In this review, we compile the literature describing recent advances in research on dedifferentiation, with an emphasis on tissue-specific findings, cellular mechanisms, and potential therapeutic applications from an engineering perspective. A critical understanding of such knowledge may provide fresh insights for designing new therapeutic strategies for regenerative medicine based on the principle of cell dedifferentiation.

Enhanced chondrogenesis in a coculture system with genetically manipulated dedifferentiated chondrocytes and ATDC5 cells.

Articular cartilage repair after injury is a great challenge worldwide due to its nerveless and avascular features. Tissue engineering is proposed as a promising alternative for cartilage regeneration. In this study, an adenoviral vector carrying the transforming growth factor-ß3 (TGF-ß3) gene was constructed and introduced into dedifferentiated chondrocytes, which were then cocultured with ATDC5 cells in an alginate hydrogel system. The results showed that the experimental groups exhibited better cell viability and higher levels of cartilage-related genes than the control groups. In this coculture system, the chondrogenic differentiation of ATDC5 cells was effectively induced by TGF-ß3 and other latent cytokines that were produced by the transfected chondrocytes. Thus, this method can avoid the degradation of exogenous TGF-ß3, and it can protect ATDC5 cells during virus transfection to maintain cell viability and chondrogenic differentiation capability. Taken together, this study provides fresh insights for applying this genetically manipulated coculture system to cartilage repair in the future.

Effects of inflammatory cytokines on bone/cartilage repair.

Many inflammatory factors can affect cell behaviors and work as a form of inter-regulatory networks through the inflammatory pathway. Inflammatory cytokines are critical for triggering bone regeneration after fracture or bone injury. Also, inflammatory cytokines play an important role in cartilage repair. The synergistic or antagonistic effects of both proinflammatory and anti-inflammatory cytokines have a great influence on fracture healing. This review discusses key inflammatory cytokines and signaling pathways involved in bone or cartilage repair.

Progress of co-culture systems in cartilage regeneration.

INTRODUCTION: Cartilage tissue engineering has rapidly developed in recent decades, exhibiting promising potential to regenerate and repair cartilage. However, the origin of a large amount of a suitable seed cell source is the major bottleneck for the further clinical application of cartilage tissue engineering. The use of a monoculture of passaged chondrocytes or mesenchymal stem cells results in undesired outcomes, such as fibrocartilage formation and hypertrophy. In the last two decades, co-cultures of chondrocytes and a variety of mesenchymal stem cells have been intensively investigated in vitro and in vivo, shedding light on the perspective of co-culture in cartilage tissue engineering. AREAS COVERED: We summarize the recent literature on the application of heterologous cell co-culture systems in cartilage tissue engineering and compare the differences between direct and indirect co-culture systems as well as discuss the underlying mechanisms. EXPERT OPINION: Co-culture system is proven to address many issues encountered by monocultures in cartilage tissue engineering, including reducing the number of chondrocytes needed and alleviating the dedifferentiation of chondrocytes. With the further development and knowledge of biomaterials, cartilage tissue engineering that combines the co-culture system and advanced biomaterials is expected to solve the difficult problem regarding the regeneration of functional cartilage.

Effect of Various Ratios of Co-Cultured ATDC5 Cells and Chondrocytes on the Expression of Cartilaginous Phenotype in Microcavitary Alginate Hydrogel.

The present study introduced a direct co-culture of mouse ATDC5 cells and primary porcine chondrocytes into a microcavitary hydrogel, which possessed advantages in promoting the growth of chondrocytes and retaining the phenotype. These two types of cells were encapsulated with gelatin microspheres in alginate hydrogels in either of the three ratios (3:1, 1:1, or 1:3 of ATDC5 cells to chondrocytes) and cultured in chondrogenic medium for 28 days. Simultaneously, the single encapsulation of ATDC5 cells or chondrocytes was set as a control. Cell Counting Kit-8 (CCK-8), real-time PCR, and immunohistochemistry staining were used to evaluate the effect of various ratios of co-cultured ATDC5 cells and chondrocytes on the expression of the cartilaginous phenotype. The CCK-8 data indicated that the ratio of 3:1 group had an outstanding ability of cell growth. The other results demonstrated that higher the ATDC5 ratios and longer the culture duration, greater the expression of cartilage-specific genes (including type II collagen and aggrecan) and more the synthesized cartilaginous extracellular matrix. Also, the Western blot analysis suggested that p44/42 MAP Kinase was involved in cell proliferation. However, due to the direct co-culture of the two cell types, the underlying mechanism necessitates further investigation. Overall, the co-culture system in microcavitary hydrogel improved the effect of chondrogenesis and exhibited promising strategy for cartilage tissue engineering therapies. J. Cell. Biochem. 118: 3607-3615, 2017. © 2017 Wiley Periodicals, Inc.

Differential circRNA expression profiles during the BMP2-induced osteogenic differentiation of MC3T3-E1 cells.

OBJECTIVE: Recent studies have indicated that circular RNAs (circRNAs) might play important roles in various diseases. However, little is known about the functions of circRNAs in the skeletal system, and the role of circRNAs in the mechanism by which bone morphogenetic protein 2 (BMP2) promotes bone differentiation remains unknown. Here, we performed RNA-seq to analyze differential expression of circRNA during different osteoblast differentiation stages and investigated the relevant mechanisms. MATERIALS AND METHODS: Alkaline phosphatase (ALP) staining and activity were performed to assess osteogenic differentiation in MC3T3-E1 cells. The expression of osteogenic markers in MC3T3-E1 cells and the differential expression levels of circRNAs were measured and validated by qRT-PCR. Osteogenic marker proteins were measured by western blot. RNA-seq was performed to detect differential expression of circRNAs during the osteogenic differentiation of MC3T3-E1 cells induced by BMP2. Gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and PANTHER pathway analyses were performed to predict the functions of differentially expressed circRNAs and potentially co-expressed target genes. The microRNA (miRNA) targets of the circRNAs and circRNA-miRNA interactions were predicted by miRanda. The circRNA-miRNA co-expression network was constructed based on the correlation analysis between the differentially expressed circRNAs and miRNAs. A graph of the circRNA-miRNA network was created using Cytoscape 3.01. RESULTS: The Cell Counting Kit 8 (CCK-8) assay showed that BMP2 promoted the proliferation of osteoblasts in vitro. Both the intracellular ALP content and activity were increased in BMP2-treated MC3T3-E1 cells. In addition, the mRNA and protein levels of the osteoblastic markers ALP, Sp7 transcription factor (SP7) and runt-related transcription factor 2 (RUNX2) were substantially up-regulated. In the present study, 158 circRNAs were differentially expressed by a fold-change ≥2.0, P<0.05 and false discovery rate <0.05. Among these, 74 circRNAs were up-regulated, while 84 circRNAs were down-regulated. In addition, the expression levels of circRNA.5846, circRNA.19142 and circRNA.10042 were significantly increased in the BMP2 group. Furthermore, by analyzing the target mRNAs of miR-7067-5p using GO and PANTHER pathway analyses, circ19142 and circ5846 were found to be not only strongly associated with the biological process of the positive regulation of developmental processes but also related to the fibroblast growth factor, epidermal growth factor, platelet-derived growth factor and Wnt signaling pathways, which are involved in cell growth and differentiation. CONCLUSION: The present study identified circ19142 and circ5846 as being associated with osteoblast differentiation and BMP2 may induce osteogenic differentiation through a circ19142/circ5846-targeted miRNA-mRNA axis.

Carboxymethylcellulose with phenolic hydroxyl microcapsules enclosinggene-modified BMSCs for controlled BMP-2 release in vitro.

OBJECTIVES: The present study aimed to develop microparticles of phenolic hydroxyl derivative of carboxymethylcellulose (CMC-Ph) via Co-flow microfluidics technology and encapsulated gene-modified rat bone mesenchymal stem cells (BMSCs) for the detection of the growth factor release was controlled by Tet-on system. Meanwhile, we investigated the effect of the CMC-Ph microcapsules and Lentiviral transduction on osteogenesis of BMP2-BMSCs. METHODS: The middle size of CMC-Ph microcapsules was prepared by optimized co-flow microfluidics through ejecting fluid CMC-Ph suspension (mixed with HRP) into co-flowing liquid paraffin which blends H2O2 at priority. The Lentivirus-encoding hBMP-2 and Tet-On system were constructed and amplified by RT-PCR, then encapsulated in the microcapsules. The cellular viability of CMC-Ph microparticles was assessed by Live/dead staining and metabolic activity was estimated by colorimetric assay kit. In addition, BMP-2 secretion and kinetic studies were determined by ELISA, alkaline phosphatase (ALP) activity was evaluated using ALP assay kit, and ALP staining as well as mineral calcium deposition was detected by alizarin red S staining. KEY FINDINGS: The diameter of CMC-Ph microparticles was controlled between 100 and 150 µm by altering the flow speed of liquid paraffin and then encapsulated bone morphogenetic protein 2 (BMP-2) gene modified BMSCs transduced by a lentiviral vector. Moreover, the mitochondrial activity of the encapsulated cells was maintained at least 24 d and BMP-2 protein secretion into the supernatant sustained for 35 d without significant loss of efficiency under the induction of the doxycycline. Furthermore, mineral deposition staining and ALP activity detection showed that encapsulated lentiviral-BMP2 transduced BMSCs possess more osteogenic differentiation potential than normal cells. CONCLUSIONS: Co-flow microfluidics and phenolic hydroxyl derivative of carboxymethylcellulose (CMC-Ph) provide a promising strategy for cell-enclosed microcapsules in combination with BMP-2 gene and Tet-on system modified BMSCs and then controlled BMP-2 protein released effectively as well as promoted the osteogenic differentiation of BMSCs.

Controlling the strontium-doping in calcium phosphate microcapsules through yeast-regulated biomimetic mineralization.

Yeast cells have controllable biosorption on metallic ions during metabolism. However, few studies were dedicated to using yeast-regulated biomimetic mineralization process to control the strontium-doped positions in calcium phosphate microcapsules. In this study, the yeast cells were allowed to pre-adsorb strontium ions metabolically and then served as sacrificing template for the precipitation and calcination of mineral shell. The pre-adsorption enabled the microorganism to enrich of strontium ions into the inner part of the microcapsules, which ensured a slow-release profile of the trace element from the microcapsule. The co-culture with human marrow stromal cells showed that gene expressions of alkaline phosphatase and Collagen-I were promoted. The promotion of osteogenic differentiation was further confirmed in the 3D culture of cell-material complexes. The strategy using living microorganism as 'smart doping apparatus' to control incorporation of trace element into calcium phosphate paved a pathway to new functional materials for hard tissue regeneration.

Prospects of osteoactivin in tissue regeneration.

INTRODUCTION: Osteoactivin (OA) was first discovered in an osteopetrotic rat model using mRNA differential display a decade ago and has been studied recently. OA in bone tissue can directly or indirectly regulate the differentiation of osteoblasts by influencing cell behaviours, such as proliferation and adhesion, as well as inducing serial signal cascades, which would be of great importance in the field of tissue engineering. The results of recent studies have further demonstrated that OA plays a critical role in the differentiation and function of cells, especially in bone formation and fracture healing. Areas covered: The discovery, structure, and function of OA as well as its therapeutic potential in tissue regeneration of bone defects, kidney injury, liver damage, and muscle atrophy. Expert opinion: OA has great potential in promoting the regeneration of damaged tissues, particularly bone tissue, which is supported by a large body of data. Future studies should focus on exploring the underlying mechanism of OA as well as pursuing the ideal form of OA-related regenerative medicine.

The enhancement of chondrogenesis of ATDC5 cells in RGD-immobilized microcavitary alginate hydrogels.

In our previous work, we have developed an effective microcavitary alginate hydrogel for proliferation of chondrocytes and maintenance of chondrocytic phenotype. In present work, we investigated whether microcavitary alginate hydrogel could promote the chondrogenesis of progenitor cells. Moreover, we attempted to further optimize this system by incorporating synthetic Arg-Gly-Asp peptide. ATDC5 cells were seeded into microcavitary alginate hydrogel with or without Arg-Gly-Asp immobilization. Cell Counting Kit-8 and live/dead staining were conducted to analyze cell proliferation. Real-time polymerase chain reaction (RT-PCR), hematoxylin and eosin, and Toluidine blue O staining as well as Western blot assay was performed to evaluate the cartilaginous markers at transcriptional level and at protein level, respectively. The obtained data demonstrated that Arg-Gly-Asp-immobilized microcavitary alginate hydrogel was preferable to promote the cell proliferation. Also, Arg-Gly-Asp-immobilized microcavitary alginate hydrogel improved the expression of chondrocytic genes including Collagen II and Aggrecan when compared with microcavitary alginate hydrogel. The results suggested that microcavitary alginate hydrogel could promote the chondrogenesis. And Arg-Gly-Asp would be promising to ameliorate this culture system for cartilage tissue engineering.

Function of sustained released resveratrol on IL-1ß-induced hBMSC MMP13 secretion inhibition and chondrogenic differentiation promotion.

Metalloproteinase-13 is the major type II collagenase that directly implicates cartilage matrix destruction. Metalloproteinase-13 is inducted and activated by interleukin-1ß, which is a commonly observed proinflammatory cytokine in the joint cavity of arthritic patients. Depression of interleukin-1ß function can inhibit metalloproteinase-13 expression and protect the cartilage extracellular matrix. In this study, resveratrol release microspheres were developed, and the direct function of the released resveratrol on the interleukin-1ß was discussed. The resveratrol-loaded microspheres were fabricated using oil-in-water emulsion and solution-evaporation methods. The particle size and the encapsulation efficiency for the techniques, which used different fabrication conditions, were within 8.3-63.9 µm and 37%-82%, respectively. The effect of drug release lasted for more than 650 h in a PBS solution at 37℃. Human bone mesenchymal stem cells were chosen for cell experiments. Interleukin-1ß was used to induce an inflammatory condition. The effect of sustained resveratrol release from the microspheres on the cells' gene expression was observed using the transwell co-culturing method. The results indicated that metalloproteinase-13 mRNA expression was upregulated after interleukin-1ß induction. The released resveratrol directly inhibited the function of interleukin-1ß and thus downregulated metalloproteinase-13 mRNA expression. Moreover, the upregulation of Col2, aggrecan and Sox9 mRNA expressions, which are major chondrocyte markers, was observed after resveratrol was released into the culture medium. Resveratrol was observed to maintain the cells' chondrogenic gene expression when subject to the inflammation condition. The sustained released resveratrol inhibited interleukin-1ß-inducted metalloproteinase-13 activation and promoted chondrocyte differentiation. This drug-loading microsphere is a promising candidate for arthritis therapy.

Redifferentiation of dedifferentiated chondrocytes in a novel three-dimensional microcavitary hydrogel.

Although chondrocytes exist in native cartilage all over the body, it is still a challenge to use them as therapeutic cells for cartilage tissue engineering (TE) because of their easy dedifferentiation in in vitro culture. An improved culture system to maintain the characteristics of chondrocytes or recover their chondrocytic phenotype should be developed. In this study, we have set up an innovative microcavitary alginate hydrogel in an easy way. We compared this culture system with the conventional hydrogel and found that the microcavitary hydrogel exhibited outstanding superiorities in helping the dedifferentiated chondrocytes recover the capability for synthesizing cartilaginous extracellular matrix. In addition, we explored the correlation between chondrocyte redifferentiation in microcavitary hydrogels and changes in p38 and Erk1/2 activity. Our findings indicated that this microcavitary hydrogel would be a promising culture system to provide sufficient competent cells for cartilage regeneration and TE.