J. Riesle

Cartilage tissue engineering

The clinical goal of tissue engineering is to restore, repair, or replace damaged tissues in the body. Significant advances have been made in recent years using stem cells as a cell source for cartilage tissue engineering and reconstructive surgery applications. Embryonic stem cells have demonstrated the potential to self-renew and differentiate into a wide range of tissues including the chondrogenic lineage, depending on culture conditions. Three-dimensional scaffolds play an important role in tissue regeneration by providing attachment sites as well as bioactive signals for cells to grow and differentiate into specific lineages. The precise microenvironments required for optimal expansion or differentiation of stem cells are only beginning to emerge now, and the controlled differentiation of embryonic stem cells in tissue engineering remains a relatively unexplored field. Hydrogels are a class of polymer-based biomaterials that have been extensively utilized in tissue engineering as scaffolds. We have demonstrated that embryonic stem cells encapsulated within poly(ethylene glycol)-based (PEGDA) photopolymerizing hydrogels and cultured in an appropriate growth factor and medium conditions undergo chondrogenic differentiation with extracellular matrix deposition characteristic of neocartilage (Hwang et al., Stem Cells 24, 284-291). Another hydrogel that has been widely used for encapsulating chondrocytes in cartilage tissue engineering is alginate. This hydrogel also has potential for tissue engineering applications using stem cells. Here, we describe the three-dimensional culture of embryonic stem cell-derived embryoid bodies in hydrogels and their differentiation toward chondrogenic lineage in chemically defined chondrogenic medium in the presence of TGF-beta1 (chondrogenic inducing conditions). We also discuss various tools and assays used for characterizing the tissue-engineered cartilage.

Enhanced chondrocyte proliferation and mesenchymal stromal cells chondrogenesis in coculture pellets mediate improved cartilage formation

In this study, we aimed at investigating the interactions between primary chondrocytes and mesenchymal stem/stromal cells (MSC) accounting for improved chondrogenesis in coculture systems. Expanded MSC from human bone marrow (BM‐MSC) or adipose tissue (AT‐MSC) were cultured in pellets alone (monoculture) or with primary human chondrocytes from articular (AC) or nasal (NC) cartilage (coculture). In order to determine the reached cell number and phenotype, selected pellets were generated by combining: (i) human BM‐MSC with bovine AC, (ii) BM‐MSC from HLA‐A2+ with AC from HLA‐A2− donors, or (iii) human green fluorescent protein transduced BM‐MSC with AC. Human BM‐MSC and AC were also cultured separately in transwells. Resulting tissues and/or isolated cells were assessed immunohistologically, biochemically, cytofluorimetrically, and by RT‐PCR. Coculture of NC or AC (25%) with BM‐MSC or AT‐MSC (75%) in pellets resulted in up to 1.6‐fold higher glycosaminoglycan content than what would be expected based on the relative percentages of the different cell types. This effect was not observed in the transwell model. BM‐MSC decreased in number (about fivefold) over time and, if cocultured with chondrocytes, increased type II collagen and decreased type X collagen expression. Instead, AC increased in number (4.2‐fold) if cocultured with BM‐MSC and maintained a differentiated phenotype. Chondro‐induction in MSC–chondrocyte coculture is a robust process mediated by two concomitant effects: MSC‐induced chondrocyte proliferation and chondrocyte‐enhanced MSC chondrogenesis. The identified interactions between progenitor and mature cell populations may lead to the efficient use of freshly harvested chondrocytes for ex vivo cartilage engineering or in situ cartilage repair. J. Cell. Physiol. 227: 88–97, 2012.

Cartilage Tissue Engineering: Controversy in the Effect of Oxygen

Articular cartilage lacks the ability to repair itself and consequently defects in this tissue do not heal. Tissue engineering approaches, employing a scaffold material and cartilage producing cells (chondrocytes), hold promise for the treatment of such defects. In these strategies the limitation of nutrients, such as oxygen, during in vitro culture are of major concern and will have implications for proper bioreactor design. We recently demonstrated that oxygen gradients are indeed present within tissue engineered cartilaginous constructs. Interestingly, oxygen, besides being an essential nutrient, is also a controlling agent of developmental processes including cartilage formation. However, the specific role of oxygen in these processes is still obscure despite the recent advances in the field. In particular, the outcome of published investigations is inconsistent regarding the effect of oxygen tension on chondrocytes. Therefore, this article describes the possible roles of oxygen gradients during embryonic cartilage development and reviews the data reported on the effect of oxygen tension on in vitro chondrocyte proliferation and differentiation from a tissue engineering perspective. Furthermore, possible causes for the variance in the data are discussed. Finally, recommendations are included that may reduce the variation, resulting in more reliable and comparable data.

Expansion of Bovine Chondrocytes on Microcarriers Enhances Redifferentiation

Functional cartilage implants for orthopedic surgery or in vitro tissue evaluation can be created from expanded chondrocytes and biodegradable scaffolds. Expansion of chondrocytes in two-dimensional culture systems results in their dedifferentiation. The hallmark of this process is the switch of collagen synthesis from type II to type I. The aim of this study was to evaluate the postexpansion chondrogenic potential of microcarrier-expanded bovine articular chondrocytes in pellet cultures. A selection of microcarriers was screened for initial attachment of chondrocytes. On the basis of those results and additional selection criteria related to clinical application, Cytodex-1 microcarriers were selected for further investigation. Comparable doubling times were obtained in T-flask and microcarrier cultures. During propagation on Cytodex-1 microcarriers, cells acquired a spherical-like morphology and the presence of collagen type II was detected. Both observations are indicative of a differentiated chondrocyte. Pellet cultures of microcarrier-expanded cells showed cartilage-like morphology and staining for proteoglycans and collagen type II after 14 days. In contrast, pellets of T-flask-expanded cells had a fibrous appearance and showed abundant staining only for collagen type I. Therefore, culture of chondrocytes on microcarriers may offer useful and cost-effective cell expansion opportunities in the field of cartilage tissue engineering.

Expansion of human nasal chondrocytes on macroporous microcarriers enhances redifferentiation

Articular cartilage has a limited capacity for self-repair. To overcome this problem, it is expected that functional cartilage replacements can be created from expanded chondrocytes seeded in biodegradable scaffolds. Expansion of chondrocytes in two-dimensional culture systems often results in dedifferentiation. This investigation focuses on the post-expansion phenotype of human nasal chondrocytes expanded on macroporous gelatin CultiSpher G microcarriers. Redifferentiation was evaluated in vitro via pellet cultures in three different culture media. Furthermore, the chondrogenic potential of expanded cells seeded in polyethylene glycol terephthalate/ polybuthylene terephthalate (PEGT/PBT) scaffolds, cultured for 14 days in vitro, and subsequently implanted subcutaneously in nude mice, was assessed. Chondrocytes remained viable during microcarrier culture and yielded doubling times (1.07+/-0.14 days) comparable to T-flask expansion (1.20+/-0.36 days). Safranin-O staining from pellet culture in different media demonstrated that production of GAG per cell was enhanced by microcarrier expansion. Chondrocyte-polymer constructs with cells expanded on microcarriers contained significantly more proteoglycans after subcutaneous implantation (288.5+/-29.2 microg) than those with T-flask-expanded cells (164.0+/-28.7 microg). Total collagen content was similar between the two groups. This study suggests that macroporous gelatin microcarriers are effective matrices for nasal chondrocyte expansion, while maintaining the ability of chondrocyte differentiation. Although the exact mechanism by which chondrocyte redifferentiation is induced through microcarrier expansion has not yet been elucidated, this technique shows promise for cartilage tissue engineering approaches.

Static and dynamic fibroblast seeding and cultivation in porous PEO/PBT scaffolds

The present study aims at optimizing dermal fibroblast seeding and cultivation in Polyactive scaffolds in order to limit the biopsy size needed for autologous treatment of full-thickness skin defects and chronic wounds. Three methods for seeding and cultivation of fibroblasts in porous scaffolds were compared: dynamic seeding followed by static cultivation (DS), static seeding followed by static cultivation (SS) and dynamic seeding followed by dynamic cultivation (DD). Human dermal fibroblasts isolated from cultured explants were seeded in porous PEO/PBT (Polyactive) scaffolds. Samples were taken from 6 h to 21 days post-seeding for both histological analysis (cell distribution and extracellular matrix (ECM) formation), and quantitative cell number assay. The seeding efficiency 24 h post-seeding was 76% (±3.6%) for dynamically seeded matrices, whereas it was only 30% (±5%) for statically seeded matrices (p < 0.001). ECM formation was abundant in DS samples already at day 10, while even after 21 days ECM formation was less pronounced in SS samples. Surprisingly, cells detached from DD samples as aggregates, starting from day 10. Cell numbers as assayed quantitatively correlated with the histological results. At all timepoints cell numbers found for DS samples were significantly higher as compared to SS samples. At day 21, DS samples contained approximately twofold more cells as compared to SS and DD samples and comprised ECM consisting of collagen types I and III. Our results indicate that the combination of dynamic seeding and static cultivation assures efficient utilization of isolated fibroblasts and improved neodermis formation, thereby allowing a reduction in the skin biopsy size needed for the engineering of living skin substitute.

Rapid prototyping of anatomically shaped, tissue-engineered implants for restoring congruent articulating surfaces in small joints

Background:  Preliminary studies investigated advanced scaffold design and tissue engineering approaches towards restoring congruent articulating surfaces in small joints.

The different behaviors of skeletal muscle cells and chondrocytes on PEGT/PBT block copolymers are related to the surface properties of the substrate.

The attachment, proliferation, morphology, and differentiation of two cell types-skeletal muscle cells and chondrocytes-were investigated on different compositions of poly(ethylene glycol) and poly(butylene terephthalate) segmented block copolymers. Four weight percentages (40, 55, 60, and 70%) and two different molecular weights (300 and 1000 Da) of poly(ethylene glycol) were tested. Varying the weight percentage and molecular weight of poly(ethylene glycol) resulted in different behaviors for skeletal muscle cells and chondrocytes. The attachment of skeletal muscle was the highest (similar to tissue culture polystyrene) when copolymers containing 55 wt % of poly(ethylene glycol) were used, regardless of the poly(ethylene glycol) molecular weight. Maximum proliferation and differentiation of skeletal muscle cells was achieved when copolymers containing 55 wt % and 300 Da molecular weight of poly(ethylene glycol) were used. In contrast, the weight percentage and molecular weight of poly(ethylene glycol) had no significant effect on chondrocyte attachment and proliferation; the attached chondrocytes retained a differentiated phenotype only when a 70 wt % of poly(ethylene glycol) was used. Cell behavior was correlated with the surface properties of the copolymer films, as indicated by contact-angle measurements. These results suggest that an optimized wt % and molecular weight of poly(ethylene glycol) will be useful depending on the specific cell type.

The effect of scaffold-cell entrapment capacity and physico-chemical properties on cartilage regeneration

An important tenet in designing scaffolds for regenerative medicine consists in mimicking the dynamic mechanical properties of the tissues to be replaced to facilitate patient rehabilitation and restore daily activities. In addition, it is important to determine the contribution of the forming tissue to the mechanical properties of the scaffold during culture to optimize the pore network architecture. Depending on the biomaterial and scaffold fabrication technology, matching the scaffolds mechanical properties to articular cartilage can compromise the porosity, which hampers tissue formation. Here, we show that scaffolds with controlled and interconnected pore volume and matching articular cartilage dynamic mechanical properties, are indeed effective to support tissue regeneration by co-cultured primary and expanded chondrocyte (1:4). Cells were cultured on scaffolds in vitro for 4 weeks. A higher amount of cartilage specific matrix (ECM) was formed on mechanically matching (M) scaffolds after 28 days. A less protein adhesive composition supported chondrocytes rounded morphology, which contributed to cartilaginous differentiation. Interestingly, the dynamic stiffness of matching constructs remained approximately at the same value after culture, suggesting a comparable kinetics of tissue formation and scaffold degradation. Cartilage regeneration in matching scaffolds was confirmed subcutaneously in vivo. These results imply that mechanically matching scaffolds with appropriate physico-chemical properties support chondrocyte differentiation.

The Effect of Timing of Mechanical Stimulation on Proliferation and Differentiation of Goat Bone Marrow Stem Cells Cultured on Braided PLGA Scaffolds

Bone marrow stromal cells (BMSCs) have been shown to proliferate and produce matrix when seeded onto braided poly(L-lactide/glycolide) acid (PLGA) scaffolds. Mechanical stimulation may be applied to stimulate tissue formation during ligament tissue engineering. This study describes for the first time the effect of constant load on BMSCs seeded onto a braided PLGA scaffold. The seeded scaffolds were subjected to four different loading regimes: Scaffolds were unloaded, loaded during seeding, immediately after seeding, or 2 days after seeding. During the first 5 days, changing the mechanical environment seemed to inhibit proliferation, because cells on scaffolds loaded immediately after seeding or after a 2-day delay, contained fewer cells than on unloaded scaffolds or scaffolds loaded during seeding (p<0.01 for scaffolds loaded after 2 days). During this period, differentiation increased with the period of load applied. After day 5, differences in cell content and collagen production leveled off. After day 11, cell number decreased, whereas collagen production continued to increase. Cell number and differentiation at day 23 were independent of the timing of the mechanical stimulation applied. In conclusion, static load applied to BMSCs cultured on PLGA scaffolds allows for proliferation and differentiation, with loading during seeding yielding the most rapid response. Future research should be aimed at elucidating the biomechanical and biochemical characteristics of tissue formed by BMSCs on PLGA under mechanical stimulation.