Cartilage Replacement Materials Research Articles

Cartilage-Inspired, High-Strength, and Heat-Tolerant Lubricating Hydrogels by Macrophase Separation.

Hydrogels are considered as a potential cartilage replacement material based on their structure being similar to natural cartilage, which are of great significance in repairing cartilage defects. However, it is difficult for the existing hydrogels to combine the high load bearing and low friction properties (37 °C) of cartilage through sample methods. Herein, we report a facile and new fabrication strategy to construct the PNIPAm/EYL hydrogel by using the macrophase separation of supersaturated N-isopropylacrylamide (NIPAm) monomer solution to promote the formation of liposomes from egg yolk lecithin (EYL) and asymmetric template method. The PNIPAm/EYL hydrogels possess a relatively high compressive strength (more than 12 MPa), fracture energy (9820 J/m2), good fatigue resistance, lubricating properties, and excellent biocompatibility. Compared with the PNIPAm hydrogel, the friction coefficient (COF 0.046) of PNIPAm/EYL hydrogel is reduced by 50%. More importantly, the COF (0.056) of PNIPAm/EYL hydrogel above lower critical solution temperature (LCST) does not increase significantly, exhibiting heat-tolerant lubricity. The finite element analysis further proves that PNIPAm/EYL hydrogel can effectively disperse the applied stress and dissipate energy under load conditions. This work not only provides new insights for the design of high-strength lubricating hydrogels but also lays a foundation for the treatment of cartilage injury as a substitute material.

Vertical and Horizontal Double Gradient Design for Super‐Slippery and High‐Bearing Hydrogel Skeleton

AbstractHydrogels with excellent hydrophilicity, high water content, outstanding biocompatibility, and lubrication properties are among the promising articular cartilage replacement materials. Despite extensive research focus on lubrication of hydrogels, realization of bioinspired materials with high load‐bearing, low‐friction, extraordinary lubrication, and anti‐wear properties is challenging. Herein, robust lubrication can be realized by structuring vertical and horizontal dual gradient surfaces, with structural design in vertical direction gradually transitioning from an anisotropic tubular to a compact body to provide load‐bearing, and a surface rigid porous polymer skeleton and a filled loose lubricating polymer structural design to provide lubrication in horizontal direction. What is more, gradient transition region in vertical direction greatly weakens gradient layer interface stress to increase load‐bearing, and gradient surface in horizontal direction is constructed by interconnected surface rigid polymer skeleton containing “pockets” that house highly hydrated and mechanically fragile soft lubrication polymer to prevent removal of lubrications under high load and wear. The designed hydrogel has an extremely low coefficient of friction (COF) with hard steel (COF ≈0.0036, 50 N, 35,000 cycles) and achieves a balance between lubrication and load bearing. The work opens up an avenue for rational design of high load‐bearing and ultralow friction soft material surfaces.

Strong, tough, and elastic poly(vinyl alcohol)/polyacrylamide DN hydrogels based on the Hofmeister effect for articular cartilage replacement.

Traditional hydrogels are usually weak and brittle, which limit their application in articular cartilage replacement because cartilage is generally strong, tough, and elastic in nature. Therefore, it is highly desirable to construct hydrogels to mimic the mechanical properties of the native articular cartilage. Herein, in this work, poly(vinyl alcohol)/polyacrylamide (PVA/PAM) DN hydrogels were prepared by in situ polymerization, which were then treated with Hofmeister series ions (Cit3-, SO42-, and Cl-) to achieve H-PVA/PAM DN hydrogels. Among the three Hofmeister ions, the DN hydrogel treated with Cit3- (named PVA/PAM-Cit) showed the densest microstructure and the highest crystallinity degree. In this context, PVA/PAM-Cit exhibited a tensile strength of 18.9 ± 1.6 MPa, a compressive strength of 102.3 ± 7.9 MPa, a tensile modulus of 10.6 ± 2.1 MPa, a compressive modulus of 8.9 ± 0.8 MPa, and a roughness of 66.2 ± 4.2 MJ m-3, respectively, which were the highest strength and modulus, and the second highest toughness when compared with those of the reported PVA and PVA based DN hydrogels so far. It also showed an extreme high elasticity, which could maintain a stress of 99.2% after 500 cycles of fatigue testing. Additionally, PVA/PAM-Cit can promote the adhesion, spreading and proliferation of chondrocytes. These results verify that such a strong, tough, and elastic hydrogel could be a novel candidate material for articular cartilage replacement.

An ultra‐tough, creep‐resistant, wear‐resistant hydrogel for cartilage replacement

AbstractAs a load‐bearing tissue, natural cartilages possess a compressive stress above 50 MPa, a water content around 60%, and a friction coefficient (FC) less than 0.1, which remains difficult for synthetic materials to realize. Herein, a facile strategy was reported to achieve this goal. The hydrogel was obtained through a three‐step strategy from polyvinyl alcohol (PVA), chitosan (CS), sodium carboxymethyl cellulose (CMC‐Na), lecithin (LS), and deionized water. First, freezing–thawing the aqueous solution of PVA and CS three times generated the precursor gel. Second, immersing it in aqueous solution of CMC‐Na to form multiple linkages (hydrogen bonds, ionic interactions, and crystallize domains). Third, it was coated with LS to form a lubrication layer. Due to the synergy of dynamic interactions, the compressive stress was 115 MPa, superior to most tough hydrogels. Owing to existence of LS, the FC was 0.03, better than the requirement for artificial cartilage. Remarkably, the FC remained stable within 50,000 wear cycles. On account of the reversible break/reformation of physical linkages, the creep recovery efficiency was 93%. The raw materials are all biocompatible and the fabrication process exclude the adoption of toxic additives. All of these features make it an ideal material for cartilage replacement.

Sandwich hydrogel to realize cartilage-mimetic structures and performances from polyvinyl alcohol, chitosan and sodium hyaluronate

Developing artificial substitutes that mimic the structures and performances of natural cartilage is of great importance. However, it is challenging to integrate the high strength, excellent biocompatibility, low coefficient of friction, long-term wear resistance, outstanding swelling resistance, and osseointegration potential into one material. Herein, a sandwich hydrogel with cartilage-mimetic structures and performances was prepared to achieve this goal. The precursor hydrogel was obtained by freezing-thawing the mixture of poly vinyl alcohol, chitosan and deionized water three cycles, accompanied by soaking in sodium hyaluronate solution. The top of the precursor hydrogel was hydrophobically modified with lauroyl chloride and then loaded with lecithin, while the bottom was mineralized with hydroxyapatite. Due to the multiple linkages (crystalline domains, hydrogen bonds, and ionic interactions), the compressive stress was 71 MPa. Owing to the synergy of the hydrophobic modification and lecithin, the coefficient of friction was 0.01. Additionally, no wear trace was observed after 50,000 wear cycles. Remarkably, hydroxyapatite enabled the hydrogel osseointegration potential. The swelling ratio of the hydrogel was 0.06 g/g after soaking in simulated synovial fluid for 7 days. Since raw materials were non-toxic, the cell viability was 100 %. All of the above merits make it an ideal material for cartilage replacement.

Microgel-Modified Bilayered Hydrogels Dramatically Boosting Load-Bearing and Lubrication.

Hydrogel-based articular cartilage replacement materials are promising candidates for their potential to provide both high load-bearing capacity and low friction performance, similar to natural cartilage. Nevertheless, the design of these materials presents a significant challenge in reconciling the conflicting demands of the load-bearing capacity and lubrication. Despite extensive research in this area, there is still room for improvement in the creation of hydrogel-based materials that effectively meet these demands. Herein, a facile strategy is provided to realize simultaneously high load-bearing and low friction properties on the proposed hydrogel by modifying the surface of mechanically strong annealled PVA-PAAc hydrogel with a high hydration potential PAAm-co-PAMPS microgel. Consequently, a bilayer hydrogel with a porous surface and a compact substrate has been obtained. Compressive experiments confirmed that the bilayer hydrogel exhibited excellent mechanical strength with a compressive strength of 32.23 MPa at 90% strain. A high load-bearing (applied load up to 30 N), extremely low friction coefficiency (0.01-0.05) and excellent wear resistance (COF low to 0.03 after a 4 h test at 10 N using a steel ball as the contact pair) are successfully achieved. These findings provide new perspectives for the design of articular cartilage materials.

Migration and remodeling in cartilage replacement materials – an in‐vitro bioreactor study

AbstractThis article presents a bioreactor study that investigates the influence of mechanical stimulation on cell migration in a cartilage replacement material. The bioreactor has been designed to carry out dynamic stimulation of cell‐seeded scaffolds, and it consists of a loading mechanism, a force‐measuring device, and a biocompatible chamber. The stimulation of the samples was carried out inside a bioreactor chamber, which simulates physiological conditions that are found in vivo. Cell migration was determined using two collagen scaffolds, a part that mimics native tissue and a second cell‐free implant. The two parts were positioned in direct contact with each other, and the samples were then subjected to cyclic compression loading for a two‐week time frame. We determined the cell viability, the number of migrated cells, and the migration depth into the initially cell‐free part of the specimen. The results from the dynamic loading were compared with an unloaded control sample. The cell migration effect was determined for different collagen concentrations, which allowed us to observe the relationship between the collagen content and cellular activity. The present results were compared to results from the literature.

Super-Strong Hydrogel Composites Reinforced with PBO Nanofibers for Cartilage Replacement.

Cartilage replacement materials exhibiting a set of demanding properties such as high water content, high mechanical stiffness, low friction, and excellent biocompatibility are quite difficult to achieve. Here, poly(p-phenylene-2,6-benzobisoxazole) (PBO) nanofibers are combined with polyvinyl alcohol (PVA) to form a super-strong structure with a performance that surpasses the vast majority of previously existing hydrogels. PVA-PBO composites with water contents in the 59-76% range exhibit tensile and compressive moduli reaching 20.3 and 4.5MPa, respectively, and a coefficient of friction below 0.08. Further, they are biocompatible and support the viability of chondrocytes for 1 week, with significant improvements in cell adhesion, proliferation, and differentiation compared to PVA. The new composites can be safely sterilized by steam heat or gamma radiation without compromising their integrity and overall performance. In addition, they show potential to be used as local delivery platforms for anti-inflammatory drugs. These attractive features make PVA-PBO composites highly competitive engineered materials with remarkable potential for use in the design of load-bearing tissues. Complementary work has also revealed that these composites will be interesting alternatives in other industrial fields where high thermal and mechanical resistance are essential requirements, or which can take advantage of the pH responsiveness functionality.

On the replacement of articular cartilage: The friction of PVA hydrogel layer in hip simulator test

The present study focuses on friction evaluation of the polyvinyl alcohol (PVA) hydrogel layer, an anticipative material for cartilage replacement. The experiments were carried out in a ball-in-socket configuration using a pendulum hip simulator. The friction coefficients of ceramic-on-hydrogel pairs were compared with those of commercial implants (metal/ceramic heads vs UHMWPE, HXPE and metal/ceramic sockets). The effects of hydrogel ageing and hydration were studied, among others. The use of PVA inserts caused up to 98% reduction in friction coefficient compared to original hip pairs. The application of PVA for local or even complete cartilage replacement seems to be an outstanding opportunity in implantology.

Bilayer Hydrogels with Low Friction and High Load-Bearing Capacity by Mimicking the Oriented Hierarchical Structure of Cartilage.

Natural articular cartilages exhibit extraordinary lubricating properties and excellent load-bearing capacity based on their penetrated surface lubricated biomacromolecules and gradient-oriented hierarchical structure. Hydrogels are considered as the most promising cartilage replacement materials due to their excellent flexibility, good biocompatibility, and low friction coefficient. However, the construction of high-strength, low-friction hydrogels to mimic cartilage is still a great challenge. Here, inspired by the structure and functions of natural articular cartilage, anisotropic hydrogels with horizontal and vertical orientation structure were constructed layer by layer and bonded with each other, successfully developing a bilayer oriented heterogeneous hydrogel with a high load-bearing capacity, low friction, and excellent fatigue resistance. The bilayer hydrogel exhibited a high compressive strength of 5.21 ± 0.45 MPa and a compressive modulus of 4.06 ± 0.31 MPa due to the enhancement mechanism of the anisotropic structure within the bottom anisotropic hydrogel. Moreover, based on the synergistic effect of the high load-bearing capacity of the bottom layer and the lubrication of the surface layer, the bilayer hydrogel possesses excellent biotribological properties in hard/soft (0.032) and soft/soft (0.028) contact, which is close to that of natural cartilage. It is worth noting that the bilayer oriented heterogeneous hydrogel is able to withstand repeated loading without fatigue crack. Therefore, this work could open up a new avenue for constructing cartilage-like materials with both high strength and low friction.

Friction and wear behaviors of HXLPE, PEEK and CoCrMo against articular cartilage under cross-shear motion

The friction and wear behaviors of cartilage replacement materials against articular cartilage are considered essential characteristics that determine the function and performance of the replacement. Previous studies have often been conducted under a reciprocal linear motion, while a cross-shear motion has been shown to widely exist in joints. The difference resulting between cross-shear motion and reciprocal linear motion, and the effect of bearing surface roughness on the frictional and wear behaviors of cartilage should be further investigated. In this study, experimental tribology investigations were conducted under the cross-shear motion for articular cartilage against commonly used three replacement materials, including high cross-linked polyethylene (HXLPE), polyether ether ketone (PEEK), and cobalt-chromium molybdenum alloy (CoCrMo). The experimental results showed that there was no significant difference in friction and wear behavior between cross-shear and reciprocal linear motion. For the implants with low surface roughness, the water contact angle of the bearing surface affected the friction and wear behaviors of the cartilage, while for the implants with high rough surfaces, the surface roughness of the material affected the friction and wear behaviors of the cartilage. The damage of cartilage under cross-shear motion mainly consisted of deformations and accumulations. This phenomenon was especially evident at high roughness. Of the three bearing materials considered, PEEK was more suitable for osteochondral implants.

PVA-AAm-AG multi-network hydrogel with high mechanical strength and cell adhesion

The self-healing ability of articular cartilage is poor, and hydrogel materials are expected to be used for cartilage replacement due to their high-water content and good biocompatibility. Here, the polyvinyl alcohol-acrylamide-agarose multi-network hydrogel was proposed. The hydrogel has a compressive modulus of 1.19 MPa and a compressive strength limit of 8.87 MPa, as well as a tensile modulus of 0.63 MPa and a tensile strength limit of 1.08 MPa. The mechanical properties of hydrogel meet the requirement for cartilage replacement materials. The in vitro cell experiments indicate that the introduction of agarose improves the cell adhesion of the hydrogel, which is expected to be beneficial in the field of biomedicine.

Development of polycarbonate urethane-based materials with controlled diclofenac release for cartilage replacement.

Hydrogels are very promising human cartilage replacement materials since they are able to mimic its structure and properties. Besides, they can be used as platforms for drug delivery to reduce inflammatory postsurgical reactions. Polycarbonate urethane (PCU) has been used in orthopedic applications due to its long-term biocompatibility and bio-durability. In this work, PCU-based hydrogels with the ability to release an anti-inflammatory (diclofenac) were developed, for the first time, for such purpose. The materials were reinforced with different amounts of cellulose acetate (CA, 10%, 15%, and 25% w/w) or carbon nanotubes (CNT, 1% and 2% w/w) in order to improve their mechanical properties. Samples were characterized in terms of compressive and tensile mechanical behavior. It was found that 15% CA and 2% CNT reinforcement led to the best mechanical properties. Thus, these materials were further characterized in terms of morphology, wettability, and friction coefficient (CoF). Contrarily to CNTs, the addition of CA significantly increased the material's porosity. Both materials became more hydrophilic, and the CoF slightly increased for PCU + 15%CA. The materials were loaded by soaking with diclofenac, and drug release experiments were conducted. PCU, PCU + 15%CA and PCU + 2%CNT presented similar release profiles, being able to ensure a controlled release of DFN for at least 4 days. Finally, in vitro cytotoxicity tests using human chondrocytes were also performed and confirmed a high biocompatibility for the three studied materials.

Porous Ultrahigh Molecular Weight Polyethylene/Functionalized Activated Nanocarbon Composites with Improved Biocompatibility.

Ultrahigh molecular weight polyethylene (UHMWPE) materials have been prevalent joint replacement materials for more than 45 years because of their excellent biocompatibility and wear resistance. In this study, functionalized activated nanocarbon (FANC) was prepared by grafting maleic anhydride polyethylene onto acid-treated activated nanocarbon. A novel porous UHMWPE composite was prepared by incorporating the appropriate amount of FANC and pore-forming agents during the hot-pressing process for medical UHMWPE powder. The experimental results showed that the best prepared porous UHMWPE/FANC exhibited appropriate tensile strength, porosity, and excellent hydrophilicity, with a contact angle of 65.9°. In vitro experiments showed that the porous UHMWPE/FANC had excellent biocompatibility, which is due to its porous structure and hydrophilicity caused by FANC. This study demonstrates the potential viability for our porous UHMWPE/FANC to be used as cartilage replacement material for biomedical applications.

Water-in-Water Emulsions, Ultralow Interfacial Tension, and Biolubrication

Water-in-Water Emulsions, Ultralow Interfacial Tension, and Biolubrication

DCECM/DBM-Radmscs Composite Scaffold Acts as a Biosynthetic Keratoprosthesis Skirt with High Biocompatability for Corneal Transplantation

An ideal keratoprosthesis scaffold should have good mechanical property, high biocompatibility, suitable corrosion and wear resistance. Due to limitation of materials, the difference of tenacity between the keratoprosthesis and the cornea often causes the exposure of keratoprosthesis materials, which results in serious complications. The cartilage has the similar biological characteristics to the cornea, no blood vessels and has certain flexibility. Although using the auricular cartilage or rib cartilage to reinforce the keratoprosthesis has been proven greatly successful, it is not possible to be individually designed and idealized. It is urgent to design a biosynthetic material for cartilage replacement to deal with predicament. We prepared and combined the decellularized cartilage extracellular matrix (DCECM) and the decellularized bone matrix (DBM), added the rabbit adipose-derived mesenchymal stem cells (rADMSCs) to construct a biosynthetic keratoprosthesis skirt. We asssessed the mechanical property, cell binding efficiency, safety and cytotoxicity, biocompatability and chondrogenic or osteogenic capacity of the DCECM/DBM-rADMSCs composite scaffold. Tests revealed the DCECM/DBM scaffold has proper elasticity, tenacity, and, is safe to cells. It’s observed that the DCECM/DBM-rADMSCs composite scaffold integrated into the recipient cornea without inflammation and rejection, while transplanted in the recipient cornea for 12 months. In particular, chondrogenesis and osteogenesis are found inside and even around the scaffold. Hence, the DCECM/DBM-rADMSCs composite scaffold coule be a potential biosynthetic skirt for keratoprosthesis or a fixed supportive scaffold to reinforce the keratoprosthesis in the treatment of end-stage corneal blindness.

Chemically crosslinked PVA hydrogels for cartilage substitution

Introduction In recent decades, hydrogels have gained increasing interest as potential cartilage replacement materials. Among these, polyvinyl alcohol (PVA) hydrogels have shown to be very promising candidates due to their biocompatibility, high degree of swelling, and elastic and rubbery nature, which allows them to mimic the natural tissues [1]. The properties of PVA-based hydrogels can be tailored by adding different compounds. The main objective of this study is to investigate the effect of adding two distinct crosslinking agents to the polymeric mixture, on the microstructure, water content, hydrophilicity, and mechanical behaviour of PVA hydrogels. Materials and Methods Four types of materials were prepared using a PVA aqueous solution (7.75% w/w) containing two different crosslinking agents: glyoxal at 0.2% and 1%, and glutaraldehyde at 5.95% and 11.9% (where the percentages indicated refer to the mass of the crosslinker relative to that of the PVA). The mixtures were poured into Petri dishes and dried at 37 °C (4 days) and then at 60 °C (2 days). The surface morphology of the samples was analysed by scanning electron microscopy (SEM) after they had been lyophilised for 48 h and coated with an Au/Pd layer. To determine the equilibrium water content (EWC), samples hydrated in pure water were weighed, dried at 60 °C until reached a constant weight, and weighed again. The water contact angles were determined in hydrated samples by the captive bubble method. To evaluate the mechanical properties, compression tests were performed with a texturometer on samples placed in an aqueous medium, using a test speed of 0.1 mm/s until a force of 5 kg was reached. Results The results showed that all materials are non-porous and have a similar surface morphology. The hydrogels crosslinked with glyoxal present higher EWC values (69.1% and 69.7% for 0.2% and 1% of glyoxal, respectively), while those with glutaraldehyde have an EWC of 52.8% and 41.5% for 5.95% and 11.9% of glutaraldehyde, correspondingly. Concerning wettability, all samples are hydrophilic and exhibit a water contact angle <55°. The modulus of elasticity of materials prepared with glyoxal is lower (1.1 − 1.4 MPa) than that of those done with glutaraldehyde (6.2 − 9.3 MPa), but their toughness is higher (0.67 − 0.45 MJ/m3 vs 0.22 − 0.27 MJ/m3). For gels crosslinked with glyoxal, the dissipated energy assumes values between 17 − 22% and for samples prepared with glutaraldehyde, it is negligible. Discussion and conclusions In conclusion, the nature and amount of crosslinking can determine the properties of PVA hydrogels. Glyoxal-containing materials have a greater water absorption capacity, are less stiff, absorb more energy, and exhibit a lower elastic recovery than glutaraldehyde-containing gels. Overall, glyoxal-crosslinked hydrogels have EWC values and mechanical properties closer to those of the natural cartilage, and therefore should be preferred as potential substitutes of this tissue.

High strength polyvinyl alcohol/polyacrylic acid (PVA/PAA) hydrogel fabricated by Cold-Drawn method for cartilage tissue substitutes

Poly (vinyl alcohol) (PVA) hydrogel has been considered as promising cartilage replacement materials due to its excellent characteristics such as high water content, low frictional behavior and excellent biocompatibility. However, lack of sufficient mechanical properties and cytocompatibility are two key obstacles for PVA hydrogel to be applied as cartilage substitutes. Herein, Polyacrylic acid (PAA) has been introduced into PVA hydrogel to balance these problems. Compared with pure PVA hydrogel, PVA/PAA hydrogel has the equal excellent biocompatibility, and its cell adhesion is significantly improved. In order to further improve the mechanical properties of hydrogels, Cold-Drawn treatment of hydrogels is performed in this paper. Compared to pure 12% PVA hydrogel, 40.8‐fold, 50.8‐fold, and 46.8‐fold increase in tensile strength, tensile modulus, and toughness, respectively, which can be obtained from 12% PVA/PAA Cold-Drawn hydrogel. These biocompatible composite hydrogels have a great application potential as cartilage tissue substitutes.

Double Network Hydrogels that Mimic the Modulus, Strength, and Lubricity of Cartilage.

The development of a hydrogel-based synthetic cartilage has the potential to overcome many limitations of current chondral defect treatments. Many attempts have been made to replicate the unique characteristics of cartilage in hydrogels, but none have simultaneously achieved high modulus, strength, and toughness while maintaining the necessary hydration required for lubricity. Herein, double network (DN) hydrogels, composed of a poly(2-acrylamido-2-methylpropanesulfonic acid) (PAMPS) first network and a poly( N-isopropylacrylamide- co-acrylamide) [P(NIPAAm- co-AAm)] second network, are evaluated as a potential off-the-shelf material for cartilage replacement. While predominantly used for its thermosensitivity, PNIPAAm is employed to achieve superior mechanical properties with its thermal transition temperature tuned above the physiological range. These PNIPAAm-based DNs demonstrate a 50-fold increase in compressive strength (∼25 MPa, similar to cartilage) compared to traditional single network hydrogels while also achieving cartilage-like modulus (∼1 MPa) and hydration (∼80%). In direct comparison to healthy cartilage (porcine), these hydrogels were confirmed to not only parallel the strength, modulus, and hydration of native articular cartilage but also exhibit a 50% lower coefficient of friction (COF). The exceptional cartilage-like properties of the PAMPS/P(NIPAAm- co-AAm) DN hydrogels makes them candidates for synthetic cartilage grafts for chondral defect repair, even in load-bearing regions of the body.

Laser perforation and cell seeding improve bacterial nanocellulose as a potential cartilage implant in the in vitro cartilage punch model

Bacterial nanocellulose (BNC) shows high biocompatibility as wound dressing or dura mater, blood vessel, and cartilage implant. Three-dimensional perforation (3-D-∅) favors migration of chondrocytes into the BNC and cartilage matrix formation. Thus, the regenerative capacity of 3-D-∅ BNC implants was tested in a standardized bovine cartilage punch model. Cartilage rings containing a central defect with an outer diameter of 6 mm and an inner diameter of 2 mm were prepared from the trochlear groove (femur-patellar articulation site). Three-D-∅ BNC implants (cell-free or cell-loaded) were cultured inside the cartilage rings for up to 12 weeks. Cartilage-BNC-constructs were then investigated by histology (hematoxylin/eosin; safranin O) and immunohistology (aggrecan, collagens 1 and 2), as well as for protein content, RNA expression, and implant push-out force. Cartilage-BNC-constructs remained vital with preserved matrix integrity during culture and almost no loss of matrix-bound proteoglycan (aggrecan) or collagen 2 from ‘host’ cartilage (with very limited quantities of collagen 1). Interestingly, 3-D-∅ BNC implants displayed: (1) significantly increased superficial, but also 3-D cell seeding over time (cell-loaded significantly earlier than cell-free); (2) progressively increased aggrecan/collagen 1 and collagen 2/collagen 1 mRNA ratios, as well as aggrecan and collagen 2 protein levels; and (3) significantly increased push-out forces over time (cell-loaded). Progressively increasing cell seeding and chondrogenic differentiation suggest beginning cartilage regeneration of the 3-D-∅ BNC in this model system, and indicate an excellent potential of 3-D-∅ BNC as a cartilage replacement material. Cell-loading may favor implant performance by accelerating cell colonization.