Wouter Schuurman

Bioprinting of hybrid tissue constructs with tailorable mechanical properties

Tissue/organ printing aims to recapitulate the intrinsic complexity of native tissues. For a number of tissues, in particular those of musculoskeletal origin, adequate mechanical characteristics are an important prerequisite for their initial handling and stability, as well as long-lasting functioning. Hence, organized implants, possessing mechanical characteristics similar to the native tissue, may result in improved clinical outcomes of regenerative approaches. Using a bioprinter, grafts were constructed by alternate deposition of thermoplastic fibers and (cell-laden) hydrogels. Constructs of different shapes and sizes were manufactured and mechanical properties, as well as cell viability, were assessed. This approach yields novel organized viable hybrid constructs, which possess favorable mechanical characteristics, within the same range as those of native tissues. Moreover, the approach allows the use of multiple hydrogels and can thus produce constructs containing multiple cell types or bioactive factors. Furthermore, since the hydrogel is supported by the thermoplastic material, a broader range of hydrogel types can be used compared to bioprinting of hydrogels alone. In conclusion, we present an innovative and versatile approach for bioprinting, yielding constructs of which the mechanical stiffness provided by thermoplastic polymers can potentially be tailored, and combined specific cell placement patterns of multiple cell types embedded in a wide range of hydrogels.

Gelatin-methacrylamide hydrogels as potential biomaterials for fabrication of tissue-engineered cartilage constructs

Gelatin-methacrylamide (gelMA) hydrogels are shown to support chondrocyte viability and differentiation and give wide ranging mechanical properties depending on several cross-linking parameters. Polymer concentration, UV exposure time, and thermal gelation prior to UV exposure allow for control over hydrogel stiffness and swelling properties. GelMA solutions have a low viscosity at 37 °C, which is incompatible with most biofabrication approaches. However, incorporation of hyaluronic acid (HA) and/or co-deposition with thermoplastics allows gelMA to be used in biofabrication processes. These attributes may allow engineered constructs to match the natural functional variations in cartilage mechanical and geometrical properties.

Hyaluronic acid and dextran-based semi-IPN hydrogels as biomaterials for bioprinting.

Bioprinting is a recent technology in tissue engineering used for the design of porous constructs through layer-by-layer deposition of cell-laden material. This technology would benefit from new biomaterials that can fulfill specific requirements for the fabrication of well-defined 3D constructs, such as the preservation of cell viability and adequate mechanical properties. We evaluated the suitability of a novel semi-interpenetrating network (semi-IPN), based on hyaluronic acid and hydroxyethyl-methacrylate-derivatized dextran (dex-HEMA), to form 3D hydrogel bioprinted constructs. The rheological properties of the solutions allowed proper handling during bioprinting, whereas photopolymerization led to stable constructs of which their mechanical properties matched the wide range of mechanical strengths of natural tissues. Importantly, excellent viability was observed for encapsulated chondrocytes. The results demonstrate the suitability of hyaluronic acid/dex-HEMA semi-IPNs to manufacture bioprinted constructs for tissue engineering.

Strategies for Zonal Cartilage Repair using Hydrogels

Articular cartilage is a highly hydrated tissue with depth-dependent cellular and matrix properties that provide low-friction load bearing in joints. However, the structure and function are frequently lost and there is insufficient repair response to regenerate high-quality cartilage. Several hydrogel-based tissue-engineering strategies have recently been developed to form constructs with biomimetic zonal variations to improve cartilage repair. Modular hydrogel systems allow for systematic control over hydrogel properties, and advanced fabrication techniques allow for control over construct organization. These technologies have great potential to address many unanswered questions involved in prescribing zonal properties to tissue-engineered constructs for cartilage repair.

Zonal Chondrocyte Subpopulations Reacquire Zone-Specific Characteristics During In Vitro Redifferentiation

Background If chondrocytes from the superficial, middle, and deep zones of articular cartilage could maintain or regain their characteristic properties during in vitro culture, it would be feasible to create constructs comprising these distinctive zones. Hypothesis Zone-specific characteristics of zonal cell populations will disappear during 2-dimensional expansion but will reappear after 3-dimensional redifferentiation, independent of the culture technique used (alginate beads versus pellet culture). Study Design Controlled laboratory study. Methods Equine articular chondrocytes from the 3 zones were expanded in monolayer culture (8 donors) and subsequently redifferentiated in pellet and alginate bead cultures for up to 4 weeks. Glycosaminoglycans and DNA were quantified, along with immunohistochemical assessment of the expression of various zonal markers, including cartilage oligomeric protein (marking cells from the deeper zones) and clusterin (specifically expressed by superficial chondrocytes). Results Cell yield varied between zones, but proliferation rates did not show significant differences. Expression of all evaluated zonal markers was lost during expansion. Compared to the alginate bead cultures, pellet cultures showed a higher amount of glycosaminoglycans produced per DNA after redifferentiation. In contrast to cells in pellet cultures, cells in alginate beads regained zonal differences, as evidenced by zone-specific reappearance of cartilage oligomeric protein and clusterin, as well as significantly higher glycosaminoglycans production by cells from the deep zone compared to the superficial zone. Conclusion Chondrocytes isolated from the 3 zones of equine cartilage can restore their zone-specific matrix expression when cultured in alginate after in vitro expansion. Clinical Relevance Appreciation of the zonal differences can lead to important advances in cartilage tissue engineering. Findings support the use of hydrogels such as alginate for engineering zonal cartilage constructs.

Cartilage regeneration using zonal chondrocyte subpopulations: a promising approach or an overcomplicated strategy?

Cartilage defects heal imperfectly and osteoarthritic changes develop frequently as a result. Although the existence of specific behaviours of chondrocytes derived from various depth‐related zones in vitro has been known for over 20 years, only a relatively small body of in vitro studies has been performed with zonal chondrocytes and current clinical treatment strategies do not reflect these native depth‐dependent (zonal) differences. This is surprising since mimicking the zonal organization of articular cartilage in neo‐tissue by the use of zonal chondrocyte subpopulations could enhance the functionality of the graft. Although some research groups including our own have made considerable progress in tailoring culture conditions using specific growth factors and biomechanical loading protocols, we conclude that an optimal regime has not yet been determined. Other unmet challenges include the lack of specific zonal cell sorting protocols and limited amounts of cells harvested per zone. As a result, the engineering of functional tissue has not yet been realized and no long‐term in vivo studies using zonal chondrocytes have been described. This paper critically reviews the research performed to date and outlines our view of the potential future significance of zonal chondrocyte populations in regenerative approaches for the treatment of cartilage defects. Secondly, we briefly discuss the capabilities of additive manufacturing technologies that can not only create patient‐specific grafts directly from medical imaging data sets but could also more accurately reproduce the complex 3D zonal extracellular matrix architecture using techniques such as hydrogel‐based cell printing. Copyright

An Electrospun Degradable Scaffold Based on a Novel Hydrophilic Polyester for Tissue‐Engineering Applications

Scaffolds based on a novel functionalized polyester, pHMGCL, are electrospun and characterized morphologically and physically. In vitro degradation studies of pHMGCL films show considerable mass loss and molecular weight reduction within 70 weeks. Scaffolds composed of fibers with uniform diameter (≈ 900 nm) and with melting temperatures higher than body temperature are prepared. As an indication for the feasibility of this material for regenerative medicine approaches, articular chondrocytes are seeded onto electrospun pHMGCL scaffolds. Chondrocytes attach to the fibers and re-differentiate as demonstrated by the production of GAG and collagen type II within four weeks of in vitro culture. Hydrophilic pHMGCL scaffolds may thus be useful for tissue engineering applications.

Three‐dimensional assembly of tissue‐engineered cartilage constructs results in cartilaginous tissue formation without retainment of zonal characteristics

Articular cartilage has limited regenerative capabilities. Chondrocytes from different layers of cartilage have specific properties, and regenerative approaches using zonal chondrocytes may yield better replication of the architecture of native cartilage than when using a single cell population. To obtain high seeding efficiency while still mimicking zonal architecture, cell pellets of expanded deep zone and superficial zone equine chondrocytes were seeded and cultured in two layers on poly(ethylene glycol)‐terephthalate–poly(butylene terephthalate) (PEGT–PBT) scaffolds. Scaffolds seeded with cell pellets consisting of a 1:1 mixture of both cell sources served as controls. Parallel to this, pellets of superficial or deep zone chondrocytes, and combinations of the two cell populations, were cultured without the scaffold. Pellet cultures of zonal chondrocytes in scaffolds resulted in a high seeding efficiency and abundant cartilaginous tissue formation, containing collagen type II and glycosaminoglycans (GAGs) in all groups, irrespective of the donor (n = 3), zonal population or stratified scaffold‐seeding approach used. However, whereas total GAG production was similar, the constructs retained significantly more GAG compared to pellet cultures, in which a high percentage of the produced GAGs were secreted into the culture medium. Immunohistochemistry for zonal markers did not show any differences between the conditions. We conclude that spatially defined pellet culture in 3D scaffolds is associated with high seeding efficiency and supports cartilaginous tissue formation, but did not result in the maintenance or restoration of the original zonal phenotype. The use of pellet‐assembled constructs leads to a better retainment of newly produced GAGs than the use of pellet cultures alone. Copyright