Hydrogels For Cartilage Tissue Engineering Research Articles

Incorporation of Aggrecan in Interpenetrating Network Hydrogels to Improve Cellular Performance for Cartilage Tissue Engineering

Interpenetrating network (IPN) hydrogels were recently introduced to the cartilage tissue engineering literature, with the approach of encapsulating cells in thermally gelling agarose that is then soaked in a poly(ethylene glycol) diacrylate (PEGDA) solution, which is then photopolymerized. These IPNs possess significantly enhanced mechanical performance desirable for cartilage regeneration, potentially allowing patients to return to weight-bearing activities quickly after surgical implantation. In an effort to improve cell viability and performance, inspiration was drawn from previous studies that have elicited positive chondrogenic responses to aggrecan, the proteoglycan largely responsible for the compressive stiffness of cartilage. Aggrecan was incorporated into the IPNs in conservative concentrations (40 μg/mL), and its effect was contrasted with the incorporation of chondroitin sulfate (CS), the primary glycosaminoglycan associated with aggrecan. Aggrecan was incorporated by physical entrapment within agarose and methacrylated CS was incorporated by copolymerization with PEGDA. The IPNs incorporating aggrecan or CS exhibited over 50% viability with encapsulated chondrocytes after 6 weeks. Both aggrecan and CS improved cell viability by 15.6% and 20%, respectively, relative to pure IPNs at 6 weeks culture time. In summary, we have introduced the novel approach of including a raw material from cartilage, namely aggrecan, to serve as a bioactive signal to cells encapsulated in IPN hydrogels for cartilage tissue engineering, which led to improved performance of encapsulated chondrocytes.

Injectable in situ-forming hydrogel for cartilage tissue engineering.

Methoxy polyethylene glycol-poly(ε-caprolactone) (MPEG-PCL; MP) diblock copolymers undergo a solution-to-gel phase transition at body temperature and serve as ideal biomaterials for drug delivery and tissue engineering. Here, we examined the potential use of a chondrocyte-loaded MP solution as an injectable, in situ-forming hydrogel for cartilage regeneration. The chondrocyte-MP solution underwent a temperature-dependent solution-to-gel phase transition in vitro, as shown by an increase in viscosity from 1 cP at 20-30 °C to 1.6 × 105 cP at 37 °C. The chondrocytes readily attached to and proliferated on the MP hydrogel in vitro. The chondrocyte-MP solution transitioned to a hydrogel immediately after subcutaneous injection into mice, and formed an interconnected pore structure required to support the growth, proliferation, and differentiation of the chondrocytes. The chondrocyte-MP hydrogels formed cartilage in vivo, as shown by the histological and immunohistochemical staining of glycosaminoglycans, proteoglycans, and type II collagen, the major components of cartilage. Cartilage formation increased with hydrogel implantation time, and the expression of glycosaminoglycans, and type II collagen reached maximal levels at 6 weeks post-implantation. Collectively, these data suggest that in situ-forming chondrocyte-MP hydrogels have potential as non-invasive alternatives for tissue-engineered cartilage formation.

A tough double network hydrogel for cartilage tissue engineering.

To develop hydrogels with high mechanical strength for cell encapsulation and three-dimensional culture is a critical challenge for cartilage tissue engineering. In this study, novel double network (DN) hydrogels were fabricated and phosphate buffer solution (PBS) was used as the solvent to facilitate cell encapsulation. The tough DN hydrogels were prepared through a two-step photopolymerization using neutral oligo(2,2-dimethyltrimethylene carbonate)-poly(ethylene glycol)-oligo(2,2-dimethyltrimethylene carbonate)-diacrylate (DPD-DA) chains for the first network and methacrylated hyaluronic acid (HA-GMA) chains for the second network. The fracture stress values of the optimized and cell-laden DN hydrogels were 8.38 ± 0.67 MPa and 6.28 ± 1.26 MPa, respectively, which showed comparable mechanical strength with natural articular cartilage tissue. The live/dead cell viability assay of cell-laden DN hydrogels demonstrated the tough DN hydrogels have comparable cytocompatibility with PEG hydrogels. After long-term culture, cartilage-specific extra cellular matrix accumulated in these DN hydrogels. The results indicated the potential of the novel DN hydrogels as cartilage tissue engineering scaffolds.

Laser sintered porous polycaprolacone scaffolds loaded with hyaluronic acid and gelatin-grafted thermoresponsive hydrogel for cartilage tissue engineering

The aim of this study is to evaluate a soft/hard bi-phase scaffold for cartilage tissue engineering. Chondrocyte proliferation, glycoaminoglycan production and total collagen content are compared between laser-sintered porous polycaprolactone (PCL) scaffolds with and without a thermoresponsive hydrogel grafted with hyaluronic acid and gelatin. The in vitro results show that scaffolds loaded with hydrogel have a higher initial chondrocyte attachment than PCL scaffolds. At day 21 and 28, scaffolds loaded with hydrogel have a significantly higher glycosaminoglycan (GAG) production than PCL scaffolds alone, and total collagen content including collagen type II in the hydrogel-loaded group is three times higher than the group without hydrogel. It is concluded that the laser-sintered porous PCL scaffold has good cytocompatibility, and that the hydrogel phase is able to enhance initial chondrocytes attachment as well as GAG and collagen production of chondrocytes. This study suggests that a soft/hard bi-phase scaffold may be used for cartilage tissue engineering to enhance in vitro chondrogenesis.

A mixed co-culture of mesenchymal stem cells and transgenic chondrocytes in alginate hydrogel for cartilage tissue engineering

To regenerate articular cartilage tissue from degeneration and trauma, synovial mesenchymal stem cells (SMSCs) were used in this study as therapeutic progenitor cells to induce therapeutic chondrogenesis. To accomplish this, chondrocytes pre-transduced with adenoviral vectors carrying the transforming growth factor (TGF) β3 gene were selected as transgenic companion cells and co-cultured side-by-side with SMSCs in a 3D environment to provide chondrogenic growth factors in situ. We adopted a mixed co-culture strategy for this purpose. Transgenic delivery of TGF-β3 in chondrocytes was performed via recombinant adenoviral vectors. The mixed co-culture of SMSCs and transgenic chondrocytes was produced in alginate gel constructs. Gene expression in both SMSCs and chondrocytes were characterized. Biochemical assays in vitro and in vivo showed that release of TGF-ß3 from transgenic chondrocytes not only induced SMSC differentiation into chondrocytic cells but also preserved the chondrocytic phenotype of chondrocytes from suspected dedifferentiation. As a result, this mixed co-culture strategy in conjunction with TGF-ß3 gene delivery could be a promising approach in cartilage tissue engineering.

Injectable chitosan hyaluronic acid hydrogels for cartilage tissue engineering

Injectable cartilaginous constructs that can form gels in tissue defects have many advantages in tissue engineering applications. In this study we created an injectable hydrogel consisting of methacrylated glycol chitosan (MeGC) and hyaluronic acid (HA) by photocrosslinking with a riboflavin photoinitiator under visible light. A minimum irradiation time of 40s was required to produce stable gels for cell encapsulation with 87–90% encapsulated chondrocyte viability. Although increasing the irradiation time from 40 to 600s significantly enhanced the compressive modulus of the hydrogels up to 11 or 17kPa for MeGC or MeGC/HA, respectively, these conditions reduced the encapsulated cell viability to 60–65%. The majority of chondrocytes encapsulated in MeGC hydrogels after 300s irradiation maintained a rounded shape with a high cell viability of ∼80–87% over a 21day culture period. The incorporation of HA in MeGC hydrogels increased the proliferation and deposition of cartilaginous extracellular matrix by encapsulated chondrocytes. These findings demonstrate that MeGC/HA composite hydrogels have the potential for cartilage repair.

Microbubbles as biocompatible porogens for hydrogel scaffolds

In this study, we explored the application of lipid-shelled, gas-filled microbubbles as a method for creating on-demand microporous hydrogels for cartilage tissue engineering. The technique allowed for homogenous distribution of cells and micropores within the scaffold, increasing the absorption coefficient of large solutes (70kDa dextran) over controls in a concentration-dependent manner. The stability of the gas phase of the microbubbles depended on several factors, including the initial size distribution of the microbubble suspension, as well as the temperature and pressure during culture. Application of pressure cycles provided controlled release of the gas phase to generate fluid-filled micropores with remnant lipid. The resulting microporous agarose scaffolds were biocompatible, leading to a twofold increase in engineered cartilage properties (EY=492±42kPa for the bubble group vs. 249±49kPa for the bubble-free control group) over a 42-day culture period. Our results suggest that microbubbles offer a simple and robust method of modulating mass transfer in cell-seeded hydrogels through mild pressurization, and the methodology may be expanded in the future to include focused ultrasound for improved spatio-temporal control.

Encapsulation of adipose-derived stem cells and transforming growth factor-β1 in carrageenan-based hydrogels for cartilage tissue engineering

Tissue engineering (TE) is an emerging field for the regeneration of damaged tissues. The combination of hydrogels with stem cells and growth factors (GFs) has become a promising approach to promote cartilage regeneration. In this study, carrageenan-based hydrogels were used to encapsulate both cells and transforming growth factor-β1 (TGF-β1). The ATDC5 cell line was encapsulated to determine the cytotoxicity and the influence of polymer concentration on cell viability and proliferation. Human adipose-derived stem cells (hASCs) were encapsulated with TGF-β1 in the hydrogel networks to enhance the chondrogenic differentiation of hASCs. Specific cartilage extracellular matrix molecules expression by hASCs were observed after 14 days of cultures of the constructs under different conditions. The κ-carrageenan was found to be a suitable biomaterial for cell and GF encapsulation. The incorporation of TGF-β1 within the carrageenan-based hydrogel enhanced the cartilage differentiation of hASCs. These findings indicate that this new system for cartilage TE is very promising for injectable thermoresponsive formulation applications.

Oligo(trimethylene carbonate)–poly(ethylene glycol)–oligo(trimethylene carbonate) triblock-based hydrogels for cartilage tissue engineering

A triblock co-polymer of oligo(trimethylene carbonate)–block-poly(ethylene glycol) 20000–block-oligo(trimethylene carbonate) diacrylate (TMC20) was used as a photo-polymerizable precursor for the encapsulation of primary articular chondrocytes. The efficacy of TMC20 as a biodegradable scaffold for cartilage tissue engineering was compared with non-degradable poly(ethylene glycol) 20000 diacrylate (PEG20) hydrogel. Chondrocytes encapsulated in PEG hydrogels containing oligo(trimethylene carbonate) (OTMC) moieties underwent spontaneous aggregation during in vitro culture, which was not observed in the PEG hydrogel counterparts. The aggregation of cells was found to be dependent on the initial cell density, as well as the mesh size of the hydrogels. Similarly, cell aggregation was also found in biodegradable PEG hydrogels containing caprolactone moieties. The aggregation of cells in TMC20 hydrogels resulted in enhanced cartilage matrix production compared with their PEG20 counterparts over 3 weeks of culture. Taken together, these results indicate that PEG hydrogels containing degradable OTMC moieties promote the aggregation and biosynthetic activity of encapsulated chondrocytes, indicating their potential as scaffolds for the repair of cartilage tissue.

In VitroandIn VivoCharacterization of Nonbiomedical- and Biomedical-Grade Alginates for Articular Chondrocyte Transplantation

Alginate is a key hydrogel for cartilage tissue engineering. Here, we systematically evaluated four biomedical- and two nonbiomedical-grade alginates for their capacity to support the in vitro culture and in vivo transplantation of articular chondrocytes. Chondrocytes in all ultrapure alginates maintained high cell viability. Spheres composed of biomedical-grade, low-viscosity, high-mannuronic acid content alginate showed the lowest decrease in size over time. Biomedical-grade, low-viscosity, high-guluronic acid content alginate allowed for optimal cell proliferation. Biomedical-grade, medium-viscosity, high-mannuronic acid content alginate promoted the highest production of proteoglycans. When transplanted into osteochondral defects in the knee joint of sheep in vivo, empty spheres were progressively surrounded by a granulation tissue. In marked contrast with these observations, all alginate spheres carrying allogeneic chondrocytes were gradually invaded by a granulation tissue containing multinucleated giant cells, lymphocytes, and fibroblasts, regardless whether they were based on biomedical- or nonbiomedical-grade alginates. After 21 days in vivo, transplanted chondrocytes were either viable or underwent necrosis, and apoptosis played a minor role in their early fate. The individual characteristics of these alginates may be valuable to tailor specific experimental and clinical strategies for cartilage tissue engineering.

Biopolymer-Based Hydrogels for Cartilage Tissue Engineering

Biopolymer-Based Hydrogels for Cartilage Tissue Engineering

The effects of fixed electrical charge on chondrocyte behavior

In this study we have compared the effects of negative and positive fixed charges on chondrocyte behavior in vitro. Electrical charges have been incorporated into oligo(poly(ethylene glycol) fumarate) (OPF) using small charged monomers such as sodium methacrylate (SMA) and (2-(methacryloyloxy) ethyl)-trimethyl ammonium chloride (MAETAC) to produce negatively and positively charged hydrogels, respectively. The physical and electrical properties of the hydrogels were characterized by measuring and calculating the swelling ratio and zeta potential, respectively. Our results revealed that the properties of these OPF modified hydrogels varied according to the concentration of charged monomers. Zeta potential measurements demonstrated that the electrical properties of the OPF hydrogel surfaces changed on incorporation of SMA and MAETAC and that these changes in electrical properties were dose-dependent. Attenuated total reflectance Fourier transform infrared spectroscopy was used to determine the hydrogel surface composition. To assess the effects of surface properties on chondrocyte behavior primary chondrocytes isolated from rabbit ears were seeded as a monolayer on top of the hydrogels. We demonstrated that the cells remained viable over 7 days and began to proliferate while seeded on top of the hydrogels. Collagen type II staining was positive in all samples, however, the staining intensity was higher on negatively charged hydrogels. Similarly, glycosaminoglycan production was significantly higher on negatively charged hydrogels compared with a neutral hydrogel. Reverse transcriptase polymerase chain reaction showed up-regulation of collagen type II and down-regulation of collagen type I on the negatively charged hydrogels. These findings indicate that charge plays an important role in establishing an appropriate environment for chondrocytes and, hence, in the engineering of cartilage. Thus, further investigations into charged hydrogels for cartilage tissue engineering is merited.

Silk hydrogel for cartilage tissue engineering

Cartilage tissue engineering based on cultivation of immature chondrocytes in agarose hydrogel can yield tissue constructs with biomechanical properties comparable to native cartilage. However, agarose is immunogenic and nondegradable, and our capability to modify the structure, composition, and mechanical properties of this material is rather limited. In contrast, silk hydrogel is biocompatible and biodegradable, and it can be produced using a water-based method without organic solvents that enables precise control of structural and mechanical properties in a range of interest for cartilage tissue engineering. We observed that one particular preparation of silk hydrogel yielded cartilaginous constructs with biochemical content and mechanical properties matching constructs based on agarose. This finding and the possibility to vary the properties of silk hydrogel motivated this study of the factors underlying the suitability of hydrogels for cartilage tissue engineering. We present data resulting from a systematic variation of silk hydrogel properties, silk extraction method, gel concentration, and gel structure. Data suggest that silk hydrogel can be used as a tool for studies of the hydrogel-related factors and mechanisms involved in cartilage formation, as well as a tailorable and fully degradable scaffold for cartilage tissue engineering.

Hierarchically Designed Agarose and Poly(Ethylene Glycol) Interpenetrating Network Hydrogels for Cartilage Tissue Engineering

A new method for encapsulating cells in interpenetrating network (IPN) hydrogels of superior mechanical integrity was developed. In this study, two biocompatible materials-agarose and poly(ethylene glycol) (PEG) diacrylate-were combined to create a new IPN hydrogel with greatly enhanced mechanical performance. Unconfined compression of hydrogel samples revealed that the IPN displayed a fourfold increase in shear modulus relative to a pure PEG-diacrylate network (39.9 vs. 9.9 kPa) and a 4.9-fold increase relative to a pure agarose network (8.2 kPa). PEG and IPN compressive failure strains were found to be 71% ± 17% and 74% ± 17%, respectively, while pure agarose gels failed around 15% strain. Similar mechanical property improvements were seen when IPNs-encapsulated chondrocytes, and LIVE/DEAD cell viability assays demonstrated that cells survived the IPN encapsulation process. The majority of IPN-encapsulated chondrocytes remained viable 1 week postencapsulation, and chondrocytes exhibited glycosaminoglycan synthesis comparable to that of agarose-encapsulated chondrocytes at 3 weeks postencapsulation. The introduction of a new method for encapsulating cells in a hydrogel with enhanced mechanical performance is a promising step toward cartilage defect repair. This method can be applied to fabricate a broad variety of cell-based IPNs by varying monomers and polymers in type and concentration and by adding functional groups such as degradable sequences or cell adhesion groups. Further, this technology may be applicable in other cell-based applications where mechanical integrity of cell-containing hydrogels is of great importance.

The composition of hydrogels for cartilage tissue engineering can influence glycosaminoglycan profile

The injectable and hydrophilic nature of hydrogels makes them suitable candidates for cartilage tissue engineering. To date, a wide range of hydrogels have been proposed for articular cartilage regeneration but few studies have quantitatively compared chondrocyte behaviour and extracellular matrix (ECM) synthesis within the hydrogels. Herein we have examined the nature of ECM synthesis by chondrocytes seeded into four hydrogels formed by either temperature change, self-assembly or chemical cross-linking. Bovine articular cartilage chondrocytes were cultured for 14 days in Extracel, Pluronic F127 blended with Type II collagen, Puramatrix and Matrixhyal. The discriminatory and sensitive technique of fluorophore-assisted carbohydrate electrophoresis (FACE) was used to determine the fine detail of the glycosaminoglycans (GAG); hyaluronan and chondroitin sulphate. FACE analysis for chondroitin sulphate and hyaluronan profiles in Puramatrix closely matched that of native cartilage. For each hydrogel, DNA content, viability and morphology were assessed. Total collagen and total sulphated GAG production were measured and normalised to DNA content. Significant differences were found in total collagen synthesis. By day 14, Extracel and Puramatrix had significantly more total collagen than Matrixhyal (1.77+/-0.26 microg and 1.97+/-0.26 microg vs. 0.60+/-0.26 microg; p<0.05). sGAG synthesis occurred in all hydrogels but a significantly higher amount of sGAG was retained within Extracel at days 7 and 14 (p<0.05). In summary, we have shown that the biochemical and biophysical characteristics of each hydrogel directly or indirectly influenced ECM formation. A detailed understanding of the ECM in the development of engineered constructs is an important step in monitoring the success of cartilage regeneration strategies.

Enzymatically-crosslinked injectable hydrogels based on biomimetic dextran–hyaluronic acid conjugates for cartilage tissue engineering

Polysaccharide hybrids consisting of hyaluronic acid (HA) grafted with a dextran–tyramine conjugate (Dex–TA) were synthesized and investigated as injectable biomimetic hydrogels for cartilage tissue engineering. The design of these hybrids (denoted as HA- g-Dex–TA) is based on the molecular structure of proteoglycans present in the extracellular matrix of native cartilage. Hydrogels of HA- g-Dex–TA were rapidly formed within 2 min via enzymatic crosslinking of the tyramine residues in the presence of horseradish peroxidase and hydrogen peroxide. The gelation time, equilibrium swelling and storage modulus could be adjusted by varying the degree of substitution of tyramine residues and polymer concentration. Bovine chondrocytes incorporated in the HA- g-Dex–TA hydrogels remained viable, as shown by the Live–dead assay. Moreover, enhanced chondrocyte proliferation and matrix production were observed in the HA- g-Dex–TA hydrogels compared to Dex–TA hydrogels. These results suggest that HA- g-Dex–TA hydrogels have a high potential as injectable scaffolds for cartilage tissue engineering.

A Newly Developed Chemically Crosslinked Dextran–Poly(Ethylene Glycol) Hydrogel for Cartilage Tissue Engineering

Cartilage tissue engineering, in which chondrogenic cells are combined with a scaffold, is a cell-based approach to regenerate damaged cartilage. Various scaffold materials have been investigated, among which are hydrogels. Previously, we have developed dextran-based hydrogels that form under physiological conditions via a Michael-type addition reaction. Hydrogels can be formed in situ by mixing a thiol-functionalized dextran with a tetra-acrylated star poly(ethylene glycol) solution. In this article we describe how the degradation time of dextran-poly(ethylene glycol) hydrogels can be varied from 3 to 7 weeks by changing the degree of substitution of thiol groups on dextran. The degradation times increased slightly after encapsulation of chondrocytes in the gels. The effect of the gelation reaction on cell viability and cartilage formation in the hydrogels was investigated. Chondrocytes or embryonic stem cells were mixed in the aqueous dextran solution, and we confirmed that the cells survived gelation. After a 3-week culturing period, chondrocytes and embryonic stem cell-derived embryoid bodies were still viable and both cell types produced cartilaginous tissue. Our data demonstrate the potential of dextran hydrogels for cartilage tissue engineering strategies.

Injectable chitosan-based hydrogels for cartilage tissue engineering

Water-soluble chitosan derivatives, chitosan- graft-glycolic acid (GA) and phloretic acid (PA) (CH-GA/PA), were designed to obtain biodegradable injectable chitosan hydrogels through enzymatic crosslinking with horseradish peroxidase (HRP) and H 2O 2. CH-GA/PA polymers were synthesized by first conjugating glycolic acid (GA) to native chitosan to render the polymer soluble at pH 7.4, and subsequent modification with phloretic acid (PA). The CH-GA43/PA10 with a degree of substitution (DS, defined as the number of substituted NH 2 groups per 100 glucopyranose rings of chitosan) of GA of 43 and DS of PA of 10 showed a good solubility at pH values up to 10. Short gelation times (e.g. 10 s at a polymer concentration of 3 wt%), as recorded by the vial tilting method, were observed for the CH-GA43/PA10 hydrogels using HRP and H 2O 2. It was shown that these hydrogels can be readily degraded by lysozyme. In vitro culturing of chondrocytes in CH-GA43/PA10 hydrogels revealed that after 2 weeks the cells were viable and retained their round shape. These features indicate that CH-GA/PA hydrogels are promising as an artificial extracellular matrix for cartilage tissue engineering.

Injectable in situ forming biodegradable chitosan–hyaluronic acid based hydrogels for cartilage tissue engineering

Injectable, biodegradable scaffolds are important biomaterials for tissue engineering and drug delivery. Hydrogels derived from natural polysaccharides are ideal scaffolds as they resemble the extracellular matrices of tissues comprised of various glycosaminoglycans (GAGs). Here, we report a new class of biocompatible and biodegradable composite hydrogels derived from water-soluble chitosan and oxidized hyaluronic acid upon mixing, without the addition of a chemical crosslinking agent. The gelation is attributed to the Schiff base reaction between amino and aldehyde groups of polysaccharide derivatives. In the current work, N-succinyl-chitosan (S-CS) and aldehyde hyaluronic acid (A-HA) were synthesized for preparation of the composite hydrogels. The polysaccharide derivatives and composite hydrogels were characterized by FTIR spectroscopy. The effect of the ratio of S-CS and A-HA on the gelation time, microstructure, surface morphology, equilibrium swelling, compressive modulus, and in vitro degradation of composite hydrogels was examined. The potential of the composite hydrogel as an injectable scaffold was demonstrated by the encapsulation of bovine articular chondrocytes within the composite hydrogel matrix in vitro. The results demonstrated that the composite hydrogel supported cell survival and the cells retained chondrocytic morphology. These characteristics provide a potential opportunity to use the injectable, composite hydrogels in tissue engineering applications.

Biological response of chondrocytes to hydrogels.

Primary bovine chondrocytes were encapsulated in alginate and alginate combined with cartilage matrix extract, Cartrigel, for the purpose of cartilage tissue engineering. The cell constructs were incubated in vitro and gene expression of cartilage-specific extracellular matrix molecules was quantitated and localized with in situ hybridization with a decrease in expression observed in the alginate-Cartrigel constructs. Further understanding of cell response to scaffolds will allow rational design and development of hydrogels for cartilage tissue engineering.