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.

Functionally improved mesenchymal stem cells via nanosecond pulsed electric fields for better treatment of osteoarthritis.

Background: Numerous approaches have been utilized to optimize mesenchymal stem cells (MSCs) performance in treating osteoarthritis (OA), however, the constrained diminished activity and chondrogenic differentiation capacity impede their therapeutic efficacy. Previous investigations have successfully shown that pretreatment with nanosecond pulsed electric fields (nsPEFs) significantly enhances the chondrogenic differentiation of MSCs. Therefore, this study aims to explore nsPEFs as a strategy to improve OA therapy by enhancing MSCs' activity and chondrogenic differentiation and also investigate its potential mechanism. Methods: In this study, a million MSCs were carefully suspended within a 0.4-cm gap cuvette and subjected to five pulses of nsPEFs (100 ns at 10 kV/cm, 1 Hz), with a 1-s interval between each pulse. A control group of MSCs was maintained without nsPEFs treatment for comparative analysis. nsPEFs were applied to regulate the MSCs performance and hinder OA progresses. In order to further explore the corresponding mechanism, we examined the changes of MSCs transcriptome after nsPEF pretreatment. Finally, we studied the properties of extracellular vesicles (EVs) secreted by MSCs affected by nsPEF and the therapeutic effect on OA. Results: We found that nsPEFs pretreatment promoted MSCs migration and viability, particularly enhancing their viability temporarily in vivo, which is also confirmed by mRNA sequencing analysis. It also significantly inhibited the development of OA-like chondrocytes in vitro and prevented OA progression in rat models. Additionally, we discovered that nsPEFs pretreatment reprogrammed MSC performance by enhancing EVs production (5.77 ± 0.92 folds), and consequently optimizing their therapeutic potential. Conclusions: In conclusion, nsPEFs pretreatment provides a simple and effective strategy for improving the MSCs performance and the therapeutic effects of MSCs for OA. EVs-nsPEFs may serve as a potent therapeutic material for OA and hold promise for future clinical applications. The translational potential of this article: This study indicates that MSCs pretreated by nsPEFs greatly inhibited the development of OA. nsPEFs pretreatment will be a promising and effective method to optimize the therapeutic effect of MSCs in the future.

Plasma and synovial fluid programmed cell death 5 (PDCD5) levels are inversely associated with TNF-α and disease activity in patients with rheumatoid arthritis.

CONTEXT: Programmed cell death 5 (PDCD5), a novel apoptotic regulatory gene, has been reported to be associated with rheumatoid arthritis (RA) and osteoarthritis (OA), which can regulate the apoptosis of synoviocytes and chondrocytes cultured in vitro. OBJECTIVE: To study expression characteristic of PDCD5 in plasma and synovial fluid of RA patients, and analyze its correlation with tumor necrosis factor alpha (TNF-α) and disease activity in RA. METHODS: A total of 135 subjects were recruited into this study (44 RA patients, 46 OA patients and 45 healthy controls). PDCD5 and TNF-α concentrations in plasma and synovial fluid were analyzed by enzyme-linked immunosorbent assay. RESULTS: Plasma and synovial fluid PDCD5 concentrations were significantly elevated in RA patients. Pearson correlation analysis indicated that plasma and synovial fluid PDCD5 levels were inversely correlated with TNF-α. Moreover, plasma PDCD5 levels were also inversely correlated with C-reactive protein and erythrocyte sedimentation rate. CONCLUSION: Plasma and synovial fluid PDCD5 could be useful for monitoring the activity and progression of RA, and its abnormal expression and dysfunction may be correlated to TNF-α in RA patients.

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.

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.

Fabrication, mechanical properties, and biocompatibility of graphene-reinforced chitosan composites.

Few-layered graphene sheets, synthesized by direct current arc-discharge method using NH(3) as one of the buffer gases, were dispersed in chitosan/acetic acid solutions. FTIR and X-ray photoelectron spectroscopy showed the presence of oxygen-containing functional groups on the surface of graphene sheets that may assist the good dispersion of graphene in chitosan solution. Graphene/chitosan films were produced by solution casting method. The mechanical properties of composite films were tested by nanoindentation method. With the addition of a small amount of graphene in chitosan (0.1-0.3 wt %), the elastic modulus of chitosan increased over ∼ 200%. The biocompatibility of graphene/chitosan composite films was checked by tetrazolium-based colorimetric assays in vitro. The cell adhesion result showed that the L929 cell can adhere to and develop on the graphene/chitosan composite films as well as on pure chitosan film, indicating that graphene/chitosan composites have good biocompatibility. Because there is no metallic impurity in graphene raw materials, the time-consuming purification process for removing metal nanoparticles entrapped in carbon nanotubes is thus avoided when graphene is used to prepare biomedical materials. Graphene/chitosan composites are potential candidates as scaffold materials in tissue engineering.

A biocompatible chitosan composite containing phosphotungstic acid modified single-walled carbon nanotubes.

Surface modification of carbon nanotubes is crucial for the dispersion and interfacial adhesion of carbon nanotubes in polymer composites. Here we present a novel method to construct single-walled carbon nanotube/chitosan composites using phosphotungstic acid as an anchor reagent to modify single-walled carbon nanotubes. The most direct benefit from this method is that this modification is mild but effective: the induced defects on single-walled carbon nanotubes are negligible based on Raman and transmission electron microscopy observations; and homogeneous dispersion of single-walled carbon nanotubes in chitosan matrices and strong binding between single-walled carbon nanotubes and chitosan are achieved. Moreover, according to the results of tetrazolium-based colorimetric assays in vitro, we demonstrate that the produced phosphotungstic-acid-modified single-walled carbon nanotube/chitosan composites have good biocompatibility. Thus, our study provides a feasible route to fabricate biocompatible composites containing single-walled carbon nanotubes for potential application in bone tissue engineering.

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.

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.

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.

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.

Mechanical dissociation of human embryonic stem cell colonies by manual scraping after collagenase treatment is much more detrimental to cellular viability than is trypsinization with gentle pipetting.

Because hESC (human embryonic stem cells) are 'social cells' that require co-operative interactions and intimate physical contact with each other, it is absolutely essential to dissociate hESC colonies into cellular clumps rather than into a single-cell suspension during serial passage. The present study compared two commonly used protocols for dissociating hESC colonies. The first protocol involved mild enzymatic treatment with collagenase type IV (1 mg/ml) for approx. 5-10 min, prior to mechanical dissociation into cellular clumps through manual scraping with a plastic pipette tip. The second protocol involved a short duration of exposure (2-3 min) to low concentrations of trypsin (0.05%), followed by gentle pipetting. The MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] assay was used to compare the recovery of viable cells after dissociating hESC colonies with these two protocols, before and after conventional freeze-thawing with 10% (v/v) DMSO. Besides undifferentiated hESC, the randomly differentiated fibroblastic progenies of hESC at various passages (P0-P4), together with an immortalized cell line (CRL-1486), were also utilized to compare the two protocols. The results demonstrated that the second protocol (trypsinization with gentle pipetting) is much less detrimental to cellular viability than is the first protocol (collagenase treatment with scratching). This in turn translated to higher freeze-thaw survival rates. It is hypothesized that scratching after collagenase treatment (first protocol) somehow induces physical damage to the cells, thereby leading to a lower recovery of viable cells, both before and after freeze-thawing.

Modification of sericin-free silk fibers for ligament tissue engineering application.

Biomedical application of silk requires the removal of sericin that is the gumming material of native silk fibers. This is because sericin can elicit an adverse immune response after implantation in the human body. However, the removal of sericin causes the silk fiber to fray and weakens its structural property, making it very difficult to knit or braid them into a scaffold for ligament tissue engineering applications. The aim of this study was to replace sericin with gelatin using NDGA as a cross-linking agent to biomimic the natural structure of native silk fibers. The physical properties and biocompatibility of the modified and native silk fibers were compared by in vitro and in vivo models. The mechanical and swelling properties of sericin-free silk fibers were greatly increased after modification with gelatin. Both modified and native silk fibers were shown to be nontoxic by in vitro cytotoxicity tests. The in vivo study demonstrated that the modified silk fibers, after 4 weeks' subcutaneous implantation in rats, caused little or no inflammatory reaction as compared with native silk fibers. The superior mechanical properties and lower inflammatory potential of modified silk fibers make them a promising candidate for ligament tissue engineering applications.

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.

Efficacy of bone marrow-derived stem cells in strengthening osteoporotic bone in a rabbit model.

Osteoporosis might be due to defects in mesenchymal stem cells (MSCs) that lead to reduced proliferation and osteoblast differentiation. We hypothesized that transplantation of MSCs into sites at risk for developing osteoporotic bone could improve bone structure and biomechanics. The aim of this study was to establish an osteoporosis rabbit model by ovariectomy (OVX), characterize the autologous MSCs from the OVX rabbits, and transplant the autologous MSCs into the OVX rabbits. MSCs harvested from bone marrow of normal and OVX rabbits were culture expanded and differentiated in osteogenic medium. Phenotypes were evaluated by collagen I immunostaining, von Kossa staining, and quantitative assays of bone-specific alkaline phosphatase (B-ALP) and osteocalcin (OCN). MSCs were transfected with green fluorescence protein (GFP) and implanted in the gluteus muscle to trace their fate in vivo. Cultured autologous MSCs from OVX rabbits were constructed in calcium alginate gels and then transplanted in the distal femurs. At 4 and 8 weeks after implantation, histomorphometrical and biomechanical analyses were performed on the samples. MSCs from OVX rabbits displayed higher B-ALP activity, but had similar OCN levels as compared to those from sham rabbits. After 8 weeks of implantation, more bone apposition was found in the MSC-alginate-treated group. Histomorphometry indicated increased trabecular thickness. Histology also illustrated improved microstructures with newly formed osteoids and enhanced trabecular thickness. In addition, biomechanical testing revealed stronger stiffness in the MSC-alginate treatment group. Therefore, this study implies that transplantation of MSCs can help to strengthen osteoporotic bone in rabbits.