Electroactive Biomaterials for Facilitating Bone Defect Repair under Pathological Conditions.

Bone degeneration associated with various diseases is increasing due to rapid aging, sedentary lifestyles, and unhealthy diets. Living bone tissue has bioelectric properties critical to bone remodeling, and bone degeneration under various pathological conditions results in significant changes to these bioelectric properties. There is growing interest in utilizing biomimetic electroactive biomaterials that recapitulate the natural electrophysiological microenvironment of healthy bone tissue to promote bone repair. This review first summarizes the etiology of degenerative bone conditions associated with various diseases such as type II diabetes, osteoporosis, periodontitis, osteoarthritis, rheumatoid arthritis, osteomyelitis, and metastatic osteolysis. Next, the diverse array of natural and synthetic electroactive biomaterials with therapeutic potential are discussed. Putative mechanistic pathways by which electroactive biomaterials can mitigate bone degeneration are critically examined, including the enhancement of osteogenesis and angiogenesis, suppression of inflammation and osteoclastogenesis, as well as their anti-bacterial effects. Finally, the limited research on utilization of electroactive biomaterials in the treatment of bone degeneration associated with the aforementioned diseases are examined. Previous studies have mostly focused on using electroactive biomaterials to treat bone traumatic injuries. It is hoped that this review will encourage more research efforts on the use of electroactive biomaterials for treating degenerative bone conditions.

Macrophages promote cartilage regeneration in a time- and phenotype-dependent manner.

Immune regulation of osteochondral defect regeneration has not yet been rigorously characterized. Although macrophages have been demonstrated to regulate the regeneration process in various tissues, their direct contribution to cartilage regeneration remains to be investigated, particularly the functions of polarized macrophage subpopulations. In this study, we investigated the origins and functions of macrophages during healing of osteochondral injury in the murine model. Upon osteochondral injury, joint macrophages are predominantly derived from circulating monocytes. Macrophages are essential for spontaneous cartilage regeneration in juvenile C57BL/6 mice, by modulating proliferation and apoptosis around the injury site. Exogeneous macrophages also exhibit therapeutic potential in promoting cartilage regeneration in adult mice with poor regenerative capacity, possibly via regulation of PDGFRα+  stem cells, with this process being influenced by initial phenotype and administration timing. Only M2c macrophages are able to promote regeneration of both cartilage tissues and subchondral bone. Overall, we reveal the direct link between macrophages and osteochondral regeneration and highlight the key roles of relevant immunological niches in successful regeneration.

Nanosecond pulsed electric fields prime mesenchymal stem cells to peptide ghrelin and enhance chondrogenesis and osteochondral defect repair in vivo.

Mesenchymal stem cells (MSCs) are important cell sources in cartilage tissue development and homeostasis, and multiple strategies have been developed to improve MSCs chondrogenic differentiation with an aim of promoting cartilage regeneration. Here we report the effects of combining nanosecond pulsed electric fields (nsPEFs) followed by treatment with ghrelin (a hormone that stimulates release of growth hormone) to regulate chondrogenesis of MSCs. nsPEFs and ghrelin were observed to separately enhance the chondrogenesis of MSCs, and the effects were significantly enhanced when the bioelectric stimulation and hormone were combined, which in turn improved osteochondral tissue repair of these cells within Sprague Dawley rats. We further found that nsPEFs can prime MSCs to be more receptive to subsequent stimuli of differentiation by upregulated Oct4/Nanog and activated JNK signaling pathway. Ghrelin initiated chondrogenic differentiation by activation of ERK1/2 signaling pathway, and RNA-seq results indicated 243 genes were regulated, and JAK-STAT signaling pathway was involved. Interestingly, the sequential order of applying these two stimuli is critical, with nsPEFs pretreatment followed by ghrelin enhanced chondrogenesis of MSCs in vitro and subsequent cartilage regeneration in vivo, but not vice versa. This synergistic prochondrogenic effects provide us new insights and strategies for future cell-based therapies.

Key considerations on the development of biodegradable biomaterials for clinical translation of medical devices: With cartilage repair products as an example.

With the interdisciplinary convergence of biology, medicine and materials science, both research and clinical translation of biomaterials are progressing at a rapid pace. However, there is still a huge gap between applied basic research on biomaterials and their translational products - medical devices, where two significantly different perspectives and mindsets often work independently and non-synergistically, which in turn significantly increases financial costs and research effort. Although this gap is well-known and often criticized in the biopharmaceutical industry, it is gradually widening. In this article, we critically examine the developmental pipeline of biodegradable biomaterials and biomaterial-based medical device products. Then based on clinical needs, market analysis, and relevant regulations, some ideas are proposed to integrate the two different mindsets to guide applied basic research and translation of biomaterial-based products, from the material and technical perspectives. Cartilage repair substitutes are discussed here as an example. Hopefully, this will lay a strong foundation for biomaterial research and clinical translation, while reducing the amount of extra research effort and funding required due to the dissonance between innovative basic research and commercialization pipeline.

Modified hyaluronic acid hydrogels with chemical groups that facilitate adhesion to host tissues enhance cartilage regeneration.

Stable integration of hydrogel implants with host tissues is of critical importance to cartilage tissue engineering. Designing and fabricating hydrogels with high adhesive strength, stability and regeneration potential are major challenges to be overcome. This study fabricated injectable adhesive hyaluronic acid (HA) hydrogel modified by aldehyde groups and methacrylate (AHAMA) on the polysaccharide backbone with multiple anchoring mechanisms (amide bond through the dynamic Schiff base reaction, hydrogen bond and physical interpenetration). AHAMA hydrogel exhibited significantly improved durability and stability within a humid environment (at least 7 days), together with higher adhesive strength (43 KPa to skin and 52 KPa to glass), as compared to commercial fibrin glue (nearly 10 KPa) and HAMA hydrogel (nearly 20 KPa). The results showed that AHAMA hydrogel was biocompatible and could be easily and rapidly prepared in situ. In vitro cell culture experiments showed that AHAMA hydrogel could enhance proliferation (1.2-folds after 3 days) and migration (1.5-folds after 12 h) of bone marrow stem cells (BMSCs), as compared to cells cultured in a culture dish. Furthermore, in a rat osteochondral defect model, implanted AHAMA hydrogel significantly promoted integration between neo-cartilage and host tissues, and significantly improved cartilage regeneration (modified O'Driscoll histological scores of 16.0 ± 4.1 and 18.3 ± 4.6 after 4 and 12-weeks of post-implantation in AHAMA groups respectively, 12.0 ± 2.7 and 12.2 ± 2.8 respectively in HAMA groups, 9.8 ± 2.4 and 11.5 ± 2.1 respectively in untreated groups). Hence, AHAMA hydrogel is a promising adhesive biomaterial for clinical cartilage regeneration and other biomedical applications.

Nanosecond pulsed electric fields enhance mesenchymal stem cells differentiation via DNMT1-regulated OCT4/NANOG gene expression.

BACKGROUND: Multiple strategies have been proposed to promote the differentiation potential of mesenchymal stem cells (MSCs), which is the fundamental property in tissue formation and regeneration. However, these strategies are relatively inefficient that limit the application. In this study, we reported a novel and efficient strategy, nanosecond pulsed electric fields (nsPEFs) stimulation, which can enhance the trilineage differentiation potential of MSCs, and further explained the mechanism behind. METHODS: We used histological staining to screen out the nsPEFs parameters that promoted the trilineage differentiation potential of MSCs, and further proved the effect of nsPEFs by detecting the functional genes. In order to explore the corresponding mechanism, we examined the expression of pluripotency genes and the methylation status of their promoters. Finally, we targeted the DNA methyltransferase which was affected by nsPEFs. RESULTS: The trilineage differentiation of bone marrow-derived MSCs was significantly enhanced in vitro by simply pre-treating with 5 pulses of nsPEFs stimulation (energy levels as 10 ns, 20 kV/cm; 100 ns, 10 kV/cm), due to that the nsPEFs demethylated the promoters of stem cell pluripotency genes OCT4 and NANOG through instantaneous downregulation of DNA methylation transferase 1 (DNMT1), thereby increasing the expression of OCT4 and NANOG for up to 3 days, and created a treatment window period of stem cells. CONCLUSIONS: In summary, nsPEFs can enhance MSCs differentiation via the epigenetic regulation and could be a safe and effective strategy for future stem cell application.

Multiple nanosecond pulsed electric fields stimulation with conductive poly(l-lactic acid)/carbon nanotubes films maintains the multipotency of mesenchymal stem cells during prolonged in vitro culture.

Mesenchymal stem cells (MSCs) gradually lose multipotency when cultured for prolonged durations in vitro, which significantly hinders subsequent clinical applications. Nanosecond pulsed electric fields (nsPEFs) have been recently investigated to overcome this problem in our lab; however, the differentiation potency of MSCs could only be partially and transiently recovered because the nsPEFs can only be delivered to suspended cells once. Here, we develop a new strategy to apply multiple nsPEFs to adherent MSCs with conductive films to mitigate the decreasing multipotency of prolonged cultured MSCs. The poly(l-lactic acid)/graphitized-carboxylated functionalized carbon nanotubes (PLLA/CNT) films were fabricated as conductive cell culture platforms. Both single and multiple nsPEFs stimulation could significantly increase the differentiation potential of MSCs, as evidenced by upregulated expression of chondrogenic, osteogenic, and adipogenic-related gene (SOX9, RUNX2, and PPAR-γ), as well as increased production of proteoglycans, mineralized calcium, and triglycerides. Multiple nsPEFs stimulation demonstrated significant efficacy in upregulating expression of pluripotency genes of OCT4A (3.5- to 4.5-folds), NANOG (3.5- to 4.0-folds), and SOX2 (1.5- to 2.0-folds) and stably maintaining high expression of these genes for nearly 23 days. Notably, nsPEFs stimulation did not significantly shorten telomere length. In conclusion, multiple nsPEFs stimulation could effectively mitigate decreasing multipotency of MSCs during prolonged in vitro culture.

Orchestrated biomechanical, structural, and biochemical stimuli for engineering anisotropic meniscus.

Reconstruction of the anisotropic structure and proper function of the knee meniscus remains an important challenge to overcome, because the complexity of the zonal tissue organization in the meniscus has important roles in load bearing and shock absorption. Current tissue engineering solutions for meniscus reconstruction have failed to achieve and maintain the proper function in vivo because they have generated homogeneous tissues, leading to long-term joint degeneration. To address this challenge, we applied biomechanical and biochemical stimuli to mesenchymal stem cells seeded into a biomimetic scaffold to induce spatial regulation of fibrochondrocyte differentiation, resulting in physiological anisotropy in the engineered meniscus. Using a customized dynamic tension-compression loading system in conjunction with two growth factors, we induced zonal, layer-specific expression of type I and type II collagens with similar structure and function to those present in the native meniscus tissue. Engineered meniscus demonstrated long-term chondroprotection of the knee joint in a rabbit model. This study simultaneously applied biomechanical, biochemical, and structural cues to achieve anisotropic reconstruction of the meniscus, demonstrating the utility of anisotropic engineered meniscus for long-term knee chondroprotection in vivo.

Nanosecond pulsed electric fields enhanced chondrogenic potential of mesenchymal stem cells via JNK/CREB-STAT3 signaling pathway.

BACKGROUND: Nanosecond pulsed electric fields (nsPEFs) can produce more significant biological effects than traditional electric fields and have thus attracted rising attention in developing medical applications based on short pulse duration and high field strength, such as effective cancer therapy. However, little is known about their effects on the differentiation of stem cells. Furthermore, mechanisms of electric fields on chondrogenic differentiation of mesenchymal stem cells (MSCs) remain elusive, and effects of electric fields on cartilage regeneration need to be verified in vivo. Here, we aimed to study the effects of nsPEFs on chondrogenic differentiation of MSCs in vitro and in vivo and further to explore the mechanisms behind the phenomenon. METHODS: The effects of nsPEF-preconditioning on chondrogenic differentiation of mesenchymal stem cells (MSCs) in vitro were evaluated using cell viability, gene expression, glycosaminoglycan (sGAG) content, and histological staining, as well as in vivo cartilage regeneration in osteochondral defects of rats. Signaling pathways were investigated with protein expression and gene expression, respectively. RESULTS: nsPEF-preconditioning with proper parameters (10 ns at 20 kV/cm, 100 ns at 10 kV/cm) significantly potentiated chondrogenic differentiation capacity of MSCs with upregulated cartilaginous gene expression and increased matrix deposition through activation of C-Jun NH2-terminal kinase (JNK) and cAMP-response element binding protein (CREB), followed by activation of downstream signal transducer and activator of transcription (STAT3). Implantation of nsPEF-preconditioned MSCs significantly enhanced cartilage regeneration in vivo, compared with implantation of non-nsPEF-preconditioned MSCs. CONCLUSION: This study demonstrates a unique approach of nsPEF treatment to potentiate the chondrogenic ability of MSCs through activation of JNK/CREB-STAT3 that could have translational potential for MSC-based cartilage regeneration.

Enhancement of the chondrogenic differentiation of mesenchymal stem cells and cartilage repair by ghrelin.

Transforming growth factor beta (TGF-ß) is commonly utilized in chondrogenic differentiation protocols, but this often results in incomplete maturation of the derived chondrocytes. Gene expression analysis, quantitation of sulfated glycosaminoglycan and collagen, and histological staining were performed to assess the effects of ghrelin. The signaling pathways involved were investigated with inhibitors or targeted by shRNAs. Joint cavity delivery of TGF-ß with or without ghrelin, within a rat cartilage defect model was performed to evaluate the in vivo effects of ghrelin. Ghrelin dramatically enhanced gene expression levels of SOX9, ACAN, and COL II and resulted in increased synthesis of sulfated glycosaminoglycan (sGAG) and collagen in vitro. Combined treatment with TGF-ß and ghrelin synergistically enhanced the phosphorylation of ERK1/2 and DMNT3A, which accounted for increased expression of chondrogenic genes. Delivery of ghrelin in combination with TGF-ß after MSC implantation within a rat osteochondral defect model significantly enhanced de novo cartilage regeneration, as compared to delivery with TGF-ß alone. In conclusion, ghrelin could significantly enhance MSC chondrogenic differentiation in vitro and can also enhance cartilage regeneration in vivo when used in combination with TGF-ß. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1387-1397, 2019.

TGF-ß1 affinity peptides incorporated within a chitosan sponge scaffold can significantly enhance cartilage regeneration.

Growth factors, such as TGF-ß and BMPs, play key roles in the chondrogenic differentiation of mesenchymal stem cells (MSCs) and cartilage regeneration in vivo. Nevertheless, there are some technical challenges in delivering exogenous growth factors in vivo, such as burst release and loss of bioactivity. In this study, TGF-ß1 affinity peptides were incorporated within porous chitosan scaffolds to enhance cartilage regeneration. Significant upregulation of gene expression levels of Sox9, Col II and AGG during chondrogenic differentiation of MSCs in vitro, were positively correlated with increasing amounts of TGF-ß1 affinity peptides incorporated within the chitosan scaffolds. The results of ectopic implantation of scaffolds in nude mice showed that incorporation of TGF-ß1 affinity peptides and preloading of TGF-ß1 synergistically enhanced ectopic cartilage formation at both high and low cell densities. Furthermore, in a rabbit osteochondral defect model, implantation of chitosan scaffolds incorporated with TGF-ß1 affinity peptides (CHI-PEP) could significantly promote cartilage regeneration, even in the absence of exogenous growth factors and seeded cells. Notably, inflammation and cartilage degeneration were markedly alleviated in the CHI-PEP group. Hence, incorporation of TGF-ß1 affinity peptide within the chitosan sponge scaffold significantly enhanced articular cartilage regeneration.

Preconditioning of mesenchymal stromal cells toward nucleus pulposus-like cells by microcryogels-based 3D cell culture and syringe-based pressure loading system.

To precondition mesenchymal stromal/stem cells (MSCs) with mechanical stimulation may enhance cell survival and functions following implantation in load bearing environment such as nucleus pulposus (NP) in intervertebral disc (IVD). In this study, preconditioning of MSCs toward NP-like cells was achieved in previously developed poly (ethylene glycol) diacrylate (PEGDA) microcryogels (PMs) within a syringe-based three-dimensional (3D) culture system which provided a facile and cost-effective pressure loading approach. PMs loaded with alginate and MSCs could be incubated in a sealable syringe which could be air-compressed to apply pressure loading through a programmable syringe pump. Expression levels of chondrogenic marker genes SOX9, COL II, and ACAN were significantly upregulated in MSCs when pressure loading of 0.2 MPa or 0.8 MPa was implemented. Expression levels of COL I and COL X were downregulated when pressure loading was applied. In a nude mouse model, MSCs loaded in PMs mechanically stimulated for three days were subcutaneously injected using the same culture syringe. Three weeks postinjection, more proteoglycans (PGs) were deposited and more SOX9 and COL II but less COL I and COL X were stained in 0.2 MPa group. Furthermore, injectable MSCs-loaded PMs were utilized in an ex vivo rabbit IVD organ culture model that demonstrated the leak-proof function and enhanced cell retention of PMs assisted cell delivery to a load bearing environment for potential NP regeneration. This microcryogels-based 3D cell culture and syringe-based pressure loading system represents a novel method for 3D cell culture with mechanical stimulation for better function. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 507-520, 2017.

Proteomic profile of mouse fibroblasts exposed to pure magnesium extract.

Magnesium and its alloys gain wide attention as degradable biomaterials. In order to reveal the molecular mechanism of the influence of biodegradable magnesium on cells, proteomics analysis was performed in this work. After mouse fibroblasts (L929) were cultured with or without Mg degradation products (Mg-extract) for 8, 24, and 48h, changes in protein expression profiles were obtained using isobaric tags for relative and absolute quantitation (iTRAQ) coupled two dimensional liquid chromatography-tandem mass spectrometry (2D LC MS/MS). A total of 867 proteins were identified (relying on at least two peptides). Compared to the control group, 205, 282, and 217 regulated proteins were identified at 8, 24, and 48h, respectively. 65 common proteins were up or down- regulated within all the three time points, which were involved in various physiological and metabolic activities. Consistent with viability, proliferation, and cell cycle analysis, stimulated energy metabolism as well as protein synthesis pathways were discussed, indicating a possible effect of Mg-extract on L929 proliferation. Furthermore, endocytosis and focal adhesion processes were also discussed. This proteomics study uncovers early cellular mechanisms triggered by Mg degradation products and highlights the cytocompatibility of biodegradable metallic materials for biomedical applications such as stents or orthopaedic implants.

Macroporous interpenetrating network of polyethylene glycol (PEG) and gelatin for cartilage regeneration.

Poor mechanical properties hinder the application of hydrogels in cartilage tissue engineering. In this study, macroporous interpenetrating network (IPN) hydrogels of gelatin and polyethylene glycol (PEG) were fabricated for use as a functional biomaterial to support chondrocyte culture. The IPN structure enhanced mechanical properties, while the macroporous structure facilitated cell-cell interactions. The hydrogels had pore sizes around 80 µm with favorable interconnectivity, reduced volume swelling ratios, and nearly unchanged weight swelling ratios with increasing gelatin ratios. More significantly, the Young's modulus increased with increasing gelatin ratio, reaching a 5.3-fold increase (p < 0.01) in IPN-10% over that of the PEG group. Chondrocytes developed elongated and fibroblast morphologies with extensive cell-cell interaction throughout IPN hydrogels, compared with round, isolated aggregates in PEG hydrogels. The glycosaminoglycan (GAG) accumulation was significantly higher in IPN hydrogels than in PEG hydrogels at day 21 and day 28. Additionally, significantly higher gene expressions of collagen II (p < 0.01) and sox-9 (p < 0.01) were found in IPN-10% when compared with other groups. Overall, the macroporous IPN hydrogels showed strong tissue formation abilities and enhanced mechanical properties, demonstrating high potential as scaffolds for cartilage regeneration.

Probing cell-matrix interactions in RGD-decorated macroporous poly (ethylene glycol) hydrogels for 3D chondrocyte culture.

Macroporous hydrogels have shown great promise as scaffolds for cartilage engineering by facilitating nutrition transport and tissue in growth. Cell-matrix adhesion-a fundamental process in tissue engineering-has shown a profound effect on subsequent cell phenotype, extracellular matrix (ECM) accumulation, and tissue reorganization. In this study, arginine-glycine-aspartic acid (RGD) was introduced to macroporous hydrogels of poly (ethylene glycol) (PEG) to fabricate PEG-G400 (with 0.4mM RGD) and PEG-G2000 (2mM RGD) to probe the cell-matrix interactions within hydrogels. Primary chondrocytes demonstrated a slightly stretched morphology with increasing RGD concentration and PEG-G2000 hydrogels boosted cell viability, proliferation, and deposition of collagen II and GAG, in comparison to the PEG-G400 and PEG-RED groups. Results also revealed chondrocytes within the cell aggregates underwent dedifferentiation and hypertrophy within RGD incorporated hydrogels, as evidenced by the high level of gene expression of collagen I on day 14 and strong immunohistological staining of collagen X and collagen I on day 35. Evidently, a high concentration of RGD (2mM RGD) enhanced cell-matrix interactions through elevating the expression of integrin ß1 and vinculin. Thus, the integration of RGD in macroporous hydrogels with a concentration of 2 mM may be sufficient for improving cell functionality, with a slight probability of dedifferentiation and hypertrophy of chondrocytes.

Cross-talk between TGF-beta/SMAD and integrin signaling pathways in regulating hypertrophy of mesenchymal stem cell chondrogenesis under deferral dynamic compression.

The molecular mechanisms of mechanotransduction in regulating mesenchymal stem cell (MSC) chondrogenesis are not fully understood and represent an area of growing investigation. In this study, human MSC was subjected to chondrogenic differentiation in chitosan-coated poly L-lactide-co-ɛ-caprolactone scaffolds under free swelling or deferral dynamic compression conditions. The effect of deferral dynamic compression to MSC chondrogenesis and late stage hypertrophy development was investigated, and the involvement of TGF-ß/SMAD pathway and integrin ß1 signaling was analyzed. Deferral dynamic compression enhanced cartilage formation and suppressed chondrocyte hypertrophy. Differential cell morphology and cytoskeletal organization were induced under dynamic compression, together with the activation of TGF-ß/Activin/Nodal and suppression of the BMP/GDP signaling. This was accompanied by the repression of integrin/FAK/ERK signaling in the non-hypertrophic cells when compared to the free swelling samples. Inhibition studies blocking TGF-ß/Activin/Nodal signaling heightened hypertrophy, activate BMP/SMAD1/5/8 and integrin signaling, while inhibition of integrin-ECM interaction suppressed hypertrophy and activate TGF-ß/SMAD2/3 in the free-swelling samples. This study demonstrates the roles of TGF-ß/SMAD and integrin signaling, and suggests cross-talk between these two signaling pathways, in regulating the compression-driven hypertrophy development.