3D Hydrogel Scaffolds for Articular Chondrocyte Culture and Cartilage Generation

Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer cartilage tissue offer a promising solution to repair articular cartilage. To select the optimal cell source for tissue repair, it is important to develop an appropriate culture platform to systematically examine the biological and biomechanical differences in the tissue-engineered cartilage by different cell sources. Here we applied a three-dimensional (3D) biomimetic hydrogel culture platform to systematically examine cartilage regeneration potential of juvenile, adult, and osteoarthritic (OA) chondrocytes. The 3D biomimetic hydrogel consisted of synthetic component poly(ethylene glycol) and bioactive component chondroitin sulfate, which provides a physiologically relevant microenvironment for in vitro culture of chondrocytes. In addition, the scaffold may be potentially used for cell delivery for cartilage repair in vivo. Cartilage tissue engineered in the scaffold can be evaluated using quantitative gene expression, immunofluorescence staining, biochemical assays, and mechanical testing. Utilizing these outcomes, we were able to characterize the differential regenerative potential of chondrocytes of varying age, both at the gene expression level and in the biochemical and biomechanical properties of the engineered cartilage tissue. The 3D culture model could be applied to investigate the molecular and functional differences among chondrocytes and progenitor cells from different stages of normal or aberrant development.     

PCL-Based Composite Scaffold Matrices for Tissue Engineering Applications

Biomaterial-based scaffolds are important cues in tissue engineering (TE) applications. Recent advances in TE have led to the development of suitable scaffold architecture for various tissue defects. In this narrative review on polycaprolactone (PCL), we have discussed in detail about the synthesis of PCL, various properties and most recent advances of using PCL and PCL blended with either natural or synthetic polymers and ceramic materials for TE applications. Further, various forms of PCL scaffolds such as porous, films and fibrous have been discussed along with the stem cells and their sources employed in various tissue repair strategies. Overall, the present review affords an insight into the properties and applications of PCL in various tissue engineering applications.     

A Phase-Field Model for Articular Cartilage Regeneration in Degradable Scaffolds

Degradable scaffolds represent a promising solution for tissue engineering of damaged or degenerated articular cartilage which due to its avascular nature, is characterized by a low self-repair capacity. To estimate the articular cartilage regeneration process employing degradable scaffolds, we propose a mathematical model as the extension of Olson and Haider’s work (Int. J. Pure Appl. Math. 53:333–353, 2009). The simulated tissue engineering procedure consists in (i) the explant of a cylindrical sample, (ii) the removal of the inner core region, and (iii) the filling of the inner region with hydrogels, degradable scaffolds enriched with nutrients, such as oxygen and glucose. The phase-field model simulates the cartilage regeneration process at the scaffold-cartilage interface. It embeds reaction-diffusion equations, which are used to model the nutrient and regenerated extracellular matrix. The equations are solved using an unconditionally stable hybrid numerical scheme. Cartilage repair processes with full-thickness defects, which are controlled by properties of hydrogel materials and cartilage explant culture based on biological interest are observed. The implemented mathematical model shows the capability to simulate cartilage repairing processes, which can be virtually controlled evaluating hydrogel and cartilage material properties including nutrient supply and defected magnitude. In particular, the adopted methodology is able to explain the regeneration time of cartilage within hydrogel environments. With the numerical scheme, the numerical simulations are demonstrated for the potential improvement of hydrogel structures.     

Electrospun Scaffold of Collagen and Polycaprolactone Containing ZnO Quantum Dots for Skin Wound Regeneration

Nanofibers(NFs)have been widely used in tissue engineering such as wound healing.In this work,the antibacterial ZnO quantum dots(ZnO QDs)have been incorporated into the biocompatible poly(ε-caprolactone)/collagen(PCL/Col)fibrous scaffolds for wound healing.The as-fabricated PCL-Col/ZnO fibrous scaffolds exhibited good swelling,antibacterial activity,and biodegradation behaviors,which were beneficial for the applications as a wound dressing.Moreover,the PCL-Col/ZnO fibrous scaffolds showed excellent cytocompatibility for promoting cell proliferation.The resultant PCL-Col/ZnO fibrous scaffolds containing vascular endothelial growth factor(VEGF)also exhibited promoted wound-healing effect through promoting expression of transforming growth factor-β(TGF-β)and the vascular factor(CD31)in tissues in the early stages of wound healing.This new electrospun fibrous scaffolds with wound-healing promotion and antibacterial property should be convenient for treating wound healing.     

PLGA微球复合明胶组织工程支架缓释蛋白药物的研究

目的：研究PLGA微球复合明胶支架对蛋白药物的释放影响。方法：将模型蛋白BSA通过复乳法制备成缓释PLGA微球,然后将微球埋置于明胶支架中,形成担载蛋白的PLGA微球复合明胶组织工程支架。考察复合支架体外蛋白释放行为,并用MicroBCA法定量测定释放的BSA量,采用β-半乳糖苷酶催化ONPG的方法检测制备前后蛋白的活性,并与不含PLGA微球直接担载蛋白的支架做对照。结果：PLGA微球复合支架蛋白的包封率能达到73．2％,其中第一天释放20％,对蛋白活性的保持达到70％以上。结论：微球复合明胶支架可以改善一般组织工程支架蛋白药物的突释,提高蛋白药物在制剂,贮存,释放过程中的稳定性。     

自体ADSCs复合PRF修复家兔耳软骨缺损的实验研究

目的:将未诱导的自体脂肪干细胞(ADSCs)与富血小板纤维蛋白(PRF)复合,作为一种全新的软骨修复材料,探讨其对家兔耳软骨全层缺损修复的可行性.方法:取家兔10只,于每只家兔耳部做4处软骨全层缺损,随机分为A、B、C、D组,A组,作为空白对照;B组植入自体ADSCs;C组植入自体PRF;D组植入自体ADSCs与PRF的复合物.分别于术后1月、2月、3月取材,进行大体及HE染色观察,并使用IPP6.0软件对软骨生成量进行半定量分析.结果:HE染色显示,3月后,A组几乎无新生软骨生成,B、C、D三组软骨生成量依次增多,D组尤为明显.IPP6.0统计结果显示,移植物植入3月后,A组软骨缺损修复率为(1.68±0.17)％,B组为(15.4±0.91.)％,C组为(32.0±2.76)％,D组为(85.77±4.88)％.各组间有显著统计学差异,与HE染色结果相符.结论:未诱导的自体ADSC s复合自体PRF作为一种全新的软骨修复材料,可以有效的修复家兔耳软骨全层缺损,具有潜在的临床应用价值.     

Synthesis and Characterization of PVA-HA-Silk Composite Hydrogel by Orthogonal Experiment

PVA-HA-Silk composite hydrogel was synthesized with polyvinyl alcohol (PVA),nano-hydroxyapatite (HA) and natural silk by using the method of repeated freezing and thawing.A series of tests were performed to study water content,stress relaxation behavior,elastic modulus,and creep characteristics of PVA-HA-Silk composite hydrogel.Orthogonal experimental design method was used to analyze the influence degree of PVA,HA and silk (three kinds of raw materials) on mechanical properties and water content of the PVA-HA-Silk composite hydrogel to select the best material ratio according to their overall performance.The results demonstrate that the mass percentage of PVA has the greatest impact on the water content,followed by HA and silk.Compression stress-strain variation of PVA-HA-Silk composite hydrogel presents a nonlinear relationship,which proves that it is a typical viscoelastic material.Comparing the mechanical properties of 16 formulas,the formula of PVA-HA-silk composite hydrogel with mass percentage of PVA 15％,HA 2.0％ and silk 1.0％ is the best.     

Purified Human Synovium Mesenchymal Stem Cells as a Good Resource for Cartilage Regeneration

Mesenchymal stem cells (MSCs) have the ability to differentiate into a variety of lineages and to renew themselves without malignant changes, and thus hold potential for many clinical applications. However, it has not been well characterized how different the properties of MSCs are depending on the tissue source in which they resided. We previously reported a novel technique for the prospective MSC isolation from bone marrow, and revealed that a combination of cell surface markers (LNGFR and THY-1) allows the isolation of highly enriched MSC populations. In this study, we isolated LNGFR⁺ THY-1 ⁺ MSCs from synovium using flow cytometry. The results show that the synovium tissue contained a significantly larger percentage of LNGFR ⁺ THY-1 ⁺ MSCs. We examined the colony formation and differentiation abilities of bone marrow-derived MSCs (BM-MSCs) and synovium-derived MSCs (SYN-MSCs) isolated from the same patients. Both types of MSCs exhibited a marked propensity to differentiate into specific lineages. BM-MSCs were preferentially differentiated into bone, while in the SYN-MSC culture, enhanced adipogenic and chondrogenic differentiation was observed. These data suggest that the tissue from which MSCs are isolated should be tailored according to their intended clinical therapeutic application.     

Fabrication of collagen hybridized elastic PLCL for tissue engineering

Biodegradable elastic poly(l-lactide-co-ε-caprolactone) (PLCL) (50:50) copolymer was blended with collagen (0.05, 0.1 and 0.2% w/w) in an acidic dioxane solution to form a collagen/PLCL hybrid material suitable for tissue engineering applications. Stability and dispersivity of collagen on collagen/PLCL hybrid films and collagen coated PLCL films under mechanical stress were determined by a collagen release test and water contact angle measurement. Hybrid films had a higher stability than collagen-coated PLCL films. Elastic recovery as well as high interconnectivity and uniform pore morphology of the hybrid scaffolds were not affected by the collagen concentration. Fibroblasts (NIH-3T3) cell culture test was performed for cell growth and viability evaluation. Collagen concentration had little affect on the initial cell adhesion after 4 h cell culture; but after 48 h cell culture, increased cell proliferation on the hybrid films was observed. The hybrid material can be applied as a scaffold for vessel and cartilage regeneration for mechano-active tissue engineering.     

Implantation of Ferumoxides Labeled Human Mesenchymal Stem Cells in Cartilage Defects

The field of tissue engineering integrates the principles of engineering, cell biology and medicine towards the regeneration of specific cells and functional tissue. Matrix associated stem cell implants (MASI) aim to regenerate cartilage defects due to arthritic or traumatic joint injuries. Adult mesenchymal stem cells (MSCs) have the ability to differentiate into cells of the chondrogenic lineage and have shown promising results for cell-based articular cartilage repair technologies. Autologous MSCs can be isolated from a variety of tissues, can be expanded in cell cultures without losing their differentiation potential, and have demonstrated chondrogenic differentiation in vitro and in vivo^{1, 2}.In order to provide local retention and viability of transplanted MSCs in cartilage defects, a scaffold is needed, which also supports subsequent differentiation and proliferation. The architecture of the scaffold guides tissue formation and permits the extracellular matrix, produced by the stem cells, to expand. Previous investigations have shown that a 2% agarose scaffold may support the development of stable hyaline cartilage and does not induce immune responses³.Long term retention of transplanted stem cells in MASI is critical for cartilage regeneration. Labeling of MSCs with iron oxide nanoparticles allows for long-term in vivo tracking with non-invasive MR imaging techniques⁴.This presentation will demonstrate techniques for labeling MSCs with iron oxide nanoparticles, the generation of cell-agarose constructs and implantation of these constructs into cartilage defects. The labeled constructs can be tracked non-invasively with MR-Imaging.Open in a separate windowClick here to view.^{(27M, flv)}     

Effect of Transplanting Various Concentrations of a Composite of Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells and Hyaluronic Acid Hydrogel on Articular Cartilage Repair in a Rabbit Model

BackgroundMesenchymal stem cells (MSCs) are known to have therapeutic potential for cartilage repair. However, the optimal concentration of MSCs for cartilage repair remains unclear. Therefore, we aimed to explore the feasibility of cartilage repair by human umbilical cord blood-derived MSCs (hUCB-MSCs) and to determine the optimal concentrations of the MSCs in a rabbit model.MethodsOsteochondral defects were created in the trochlear groove of femur in 55 rabbits. Four experimental groups (11 rabbits/group) were treated by transplanting the composite of hUCB-MSCs and HA with various MSCs concentrations (0.1, 0.5, 1.0, and 1.5 x 10⁷ cells/ml). One control group was left untreated. At 4, 8, and 16 weeks post-transplantation, the degree of cartilage repair was evaluated grossly and histologically.FindingsOverall, transplanting hUCB-MSCs and HA hydrogel resulted in cartilage repair tissue with better quality than the control without transplantation (P = 0.015 in 0.1, P = 0.004 in 0.5, P = 0.004 in 1.0, P = 0.132 in 1.5 x 10⁷ cells/ml). Interestingly, high cell concentration of hUCB-MSCs (1.5×10⁷ cells/ml) was inferior to low cell concentrations (0.1, 0.5, and 1.0 x 10⁷ cells/ml) in cartilage repair (P = 0.394,P = 0.041, P = 0.699, respectively). The 0.5 x 10⁷ cells/ml group showed the highest cartilage repair score at 4, 8 and 16 weeks post transplantation, and followed by 0.1x10⁷ cells/ml group or 1.0 x 10⁷ cell/ml group.ConclusionsThe results of this study suggest that transplantation of the composite of hUCB-MSCs and HA is beneficial for cartilage repair. In addition, this study shows that optimal MSC concentration needs to be determined for better cartilage repair.     

Polyhydroxybutyrate/Hydroxyapatite Highly Porous Scaffold for Small Bone Defects Replacement in the Nonload-bearing Parts

In the present work,Polyhydroxybutyrate (PHB)/Hydroxyapatite (HA) porous composites (10％,20％,30 ％,40％,50％ weight HA) were obtained by sintering.PHB/20％ HA optimally combines satisfactory mechanical properties with a high content of the bioactive component (HA).Porous PHB/20％ HA scaffolds have shown high mechanical properties (compressive strength of 106 MPa and Young's modulus of 901 MPa).A high volume fraction of interconnected pores (＞ 50 vol.％) was achieved with pore size of 50 μm-500 μm.Biocompatibility of porous pure PHB and PHB/20％HA,as its osseointegration were assessed in vitro and after implantation in laboratory animals.PHB/20％ HA (-5％ ± 0.9％) and pure PHB (-3％ ± 1.4％) samples after 24 hours of incubation with human leucocytes showed no significant level of cytotoxicity when p =0.648 (p-value).In vitro massive adhesion of mouse Multipotent Mesenchymal Stromal Cells (MMSC) to the surface of both porous samples was shown.PHB/20％ HA induced more intensive MMSC proliferation compared to pure PHB,which are 31％ ± 6.1％ and 20％ ± 5.7 ％ respectively when p =0.039.We observed the resorption (implant surface area was reduced by 49 ％) and integration of the porous PHB/20％ HA samples into surrounding tissues after 30 days of implantation.The signs of osteoclasts accumulation,neo-angigenesis and new bone formation were observed,which make PHB/20％ HA promising for bone tissue engineering.     

Cranial Suture Regeneration Mitigates Skull and Neurocognitive Defects in Craniosynostosis

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Influence of a Superficial Tangential Zone Over Repairing Cartilage Defects: Implications for Tissue Engineering

The superficial tangential zone (STZ) plays a critical role in normal cartilage function but is not yet a focus for designing tissue-engineered constructs for cartilage repair. Without material properties of sufficient quality in this zone, transplanted constructs in vivo may have little chance of survival. This finite element study investigates the impact of the superficial tangential zone on the mechanical function of normal articular surfaces as well as those with transplanted constructs. The zone is modeled as a thin transversely isotropic material with strain dependent permeability. The analyses predict that a normal transversely isotropic STZ placed over a repair region reduces the axial compression (55–68%) of, and the rate of fluid loss (45–82%) from the articular surface. A reduction was also found in von Mises stress (26–57%), axial strain (22–56%), and radial strain (69–73%), and an increase in fluid pressure (19–45%) in repair tissue under the STZ. Incorporating a quality superficial tangential zone in tissue-engineered constructs may be a critical factor in achieving mechanical environments conducive for successful cartilage repairs.     

MR Imaging Features of Gadofluorine-Labeled Matrix-Associated Stem Cell Implants in Cartilage Defects

Objectives

The purpose of our study was to assess the chondrogenic potential and the MR signal effects of GadofluorineM-Cy labeled matrix associated stem cell implants (MASI) in pig knee specimen.

Materials and Methods

Human mesenchymal stem cells (hMSCs) were labeled with the micelle-based contrast agent GadofluorineM-Cy. Ferucarbotran-labeled hMSCs, non-labeled hMSCs and scaffold only served as controls. Chondrogenic differentiation was induced and gene expression and histologic evaluation were performed. The proportions of spindle-shaped vs. round cells of chondrogenic pellets were compared between experimental groups using the Fisher''s exact test. Labeled and unlabeled hMSCs and chondrocytes in scaffolds were implanted into cartilage defects of porcine femoral condyles and underwent MR imaging with T1- and T2-weighted SE and GE sequences. Contrast-to-noise ratios (CNR) between implants and adjacent cartilage were determined and analyzed for significant differences between different experimental groups using the Kruskal-Wallis test. Significance was assigned for p<0.017, considering a Bonferroni correction for multiple comparisons.

Results

Collagen type II gene expression levels were not significantly different between different groups (p>0.017). However, hMSC differentiation into chondrocytes was superior for unlabeled and GadofluorineM-Cy-labeled cells compared with Ferucarbotran-labeled cells, as evidenced by a significantly higher proportion of spindle cells in chondrogenic pellets (p<0.05). GadofluorineM-Cy-labeled hMSCs and chondrocytes showed a positive signal effect on T1-weighted images and a negative signal effect on T2-weighted images while Ferucarbotran-labeled cells provided a negative signal effect on all sequences. CNR data for both GadofluorineM-Cy-labeled and Ferucarbotran-labeled hMSCs were significantly different compared to unlabeled control cells on T1-weighted SE and T2*-weighted MR images (p<0.017).

Conclusion

hMSCs can be labeled by simple incubation with GadofluorineM-Cy. The labeled cells provide significant MR signal effects and less impaired chondrogenesis compared to Ferucarbotran-labeled hMSCs. Thus, GadoflurineM-Cy might represent an alternative MR cell marker to Ferucarbotran, which is not distributed any more in Europe or North America.     

A Novel Injectable Calcium Phosphate Cement-Bioactive Glass Composite for Bone Regeneration

Background

Calcium phosphate cement (CPC) can be molded or injected to form a scaffold in situ, which intimately conforms to complex bone defects. Bioactive glass (BG) is known for its unique ability to bond to living bone and promote bone growth. However, it was not until recently that literature was available regarding CPC-BG applied as an injectable graft. In this paper, we reported a novel injectable CPC-BG composite with improved properties caused by the incorporation of BG into CPC.

Materials and Methods

The novel injectable bioactive cement was evaluated to determine its composition, microstructure, setting time, injectability, compressive strength and behavior in a simulated body fluid (SBF). The in vitro cellular responses of osteoblasts and in vivo tissue responses after the implantation of CPC-BG in femoral condyle defects of rabbits were also investigated.

Results

CPC-BG possessed a retarded setting time and markedly better injectability and mechanical properties than CPC. Moreover, a new Ca-deficient apatite layer was deposited on the composite surface after immersing immersion in SBF for 7 days. CPC-BG samples showed significantly improved degradability and bioactivity compared to CPC in simulated body fluid (SBF). In addition, the degrees of cell attachment, proliferation and differentiation on CPC-BG were higher than those on CPC. Macroscopic evaluation, histological evaluation, and micro-computed tomography (micro-CT) analysis showed that CPC-BG enhanced the efficiency of new bone formation in comparison with CPC.

Conclusions

A novel CPC-BG composite has been synthesized with improved properties exhibiting promising prospects for bone regeneration.     

Synthesis and Characterization of Pectin/PVP Hydrogel Membranes for Drug Delivery System

The purpose of the present study was to develop and design pectin and polyvinyl pyrrolidone (PVP) blended hydrogel membranes (PEVP), with different pectin: PVP ratios (1:0.2, 1:0.4, 1:0.6, 1:0.8 and 1:1 w/w), which were prepared by using a conventional solution casting technique. An attempt has been made to characterize the hydrogel membranes by various instrumental techniques like, FTIR (Fourier transform infrared) spectroscopy, X-ray diffraction (XRD), Differential scanning calorimetry (DSC), tensile strength test and scanning electron microscopy (SEM). The release patterns of the drug (salicylic acid) from the hydrogel membrane were done in three different release mediums (pH 1.4, pH 7.4 and distilled water) and samples were analyzed spectrophotometrically at 294 nm wavelength on a UV Vis spectrophotometer. MTT assay was done to ensure cytocompatibility of the pectin/PVP hydrogel membranes using B16 melanoma cells. FTIR spectroscopy indicated the presence of secondary amide (I) absorption bands. The XRD study shows decrease in crystallinity of the hydrogel membranes with increase in PVP ratio. DSC study shows an increase in T _g of pectin after blending with PVP. It was found that tensile strength increases with increasing PVP ratios in the hydrogel membranes. The prepared hydrogel membranes were found to be biocompatible with B16 melanoma cells.