Engineering cartilaginous grafts using chondrocyte-laden hydrogels supported by a superficial layer of stem cells.

During postnatal joint development, progenitor cells that reside in the superficial region of articular cartilage first drive the rapid growth of the tissue and later help direct the formation of mature hyaline cartilage. These developmental processes may provide directions for the optimal structuring of co-cultured chondrocytes (CCs) and multipotent stromal/stem cells (MSCs) required for engineering cartilaginous tissues. The objective of this study was to engineer cartilage grafts by recapitulating aspects of joint development where a population of superficial progenitor cells drives the development of the tissue. To this end, MSCs were either self-assembled on top of CC-laden agarose gels (structured co-culture) or were mixed with CCs before being embedded in an agarose hydrogel (mixed co-culture). Porcine infrapatellar fat pad-derived stem cells (FPSCs) and bone marrow-derived MSCs (BMSCs) were used as sources of progenitor cells. The DNA, sGAG and collagen content of a mixed co-culture of FPSCs and CCs was found to be lower than the combined content of two control hydrogels seeded with CCs and FPSCs only. In contrast, a mixed co-culture of BMSCs and CCs led to increased proliferation and sGAG and collagen accumulation. Of note was the finding that a structured co-culture, at the appropriate cell density, led to greater sGAG accumulation than a mixed co-culture for both MSC sources. In conclusion, assembling MSCs onto CC-laden hydrogels dramatically enhances the development of the engineered tissue, with the superficial layer of progenitor cells driving CC proliferation and cartilage ECM production, mimicking certain aspects of developing cartilage. Copyright © 2015 John Wiley & Sons, Ltd.

Tissue Engineering Whole Bones Through Endochondral Ossification: Regenerating the Distal Phalanx.

Novel strategies are urgently required to facilitate regeneration of entire bones lost due to trauma or disease. In this study, we present a novel framework for the regeneration of whole bones by tissue engineering anatomically shaped hypertrophic cartilaginous grafts in vitro that subsequently drive endochondral bone formation in vivo. To realize this, we first fabricated molds from digitized images to generate mesenchymal stem cell-laden alginate hydrogels in the shape of different bones (the temporomandibular joint [TMJ] condyle and the distal phalanx). These constructs could be stimulated in vitro to generate anatomically shaped hypertrophic cartilaginous tissues that had begun to calcify around their periphery. Constructs were then formed into the shape of the distal phalanx to create the hypertrophic precursor of the osseous component of an engineered long bone. A layer of cartilage engineered through self-assembly of chondrocytes served as the articular surface of these constructs. Following chondrogenic priming and subcutaneous implantation, the hypertrophic phase of the engineered phalanx underwent endochondral ossification, leading to the generation of a vascularized bone integrated with a covering layer of stable articular cartilage. Furthermore, spatial bone deposition within the construct could be modulated by altering the architecture of the osseous component before implantation. These findings open up new horizons to whole limb regeneration by recapitulating key aspects of normal bone development.

Engineering cartilage or endochondral bone: a comparison of different naturally derived hydrogels.

Cartilaginous tissues engineered using mesenchymal stem cells (MSCs) have been shown to generate bone in vivo by executing an endochondral programme. This may hinder the use of MSCs for articular cartilage regeneration, but opens the possibility of using engineered cartilaginous tissues for large bone defect repair. Hydrogels may be an attractive tool in the scaling-up of such tissue engineered grafts for endochondral bone regeneration. In this study, we compared the capacity of different naturally derived hydrogels (alginate, chitosan and fibrin) to support chondrogenesis and hypertrophy of MSCs in vitro and endochondral ossification in vivo. In vitro, alginate and chitosan constructs accumulated the highest levels of sulfated glycosaminoglycan (sGAG), with chitosan constructs synthesizing the highest levels of collagen. Alginate and fibrin constructs supported the greatest degree of calcium accumulation, though only fibrin constructs calcified homogeneously. In vivo, chitosan constructs facilitated neither vascularization nor endochondral ossification, and also retained the greatest amount of sGAG, suggesting it to be a more suitable material for the engineering of articular cartilage. Both alginate and fibrin constructs facilitated vascularization and endochondral bone formation as well as the development of a bone marrow environment. Alginate constructs accumulated significantly more mineral and supported greater bone formation in central regions of the engineered tissue. In conclusion, this study demonstrates the capacity of chitosan hydrogels to promote and better maintain a chondrogenic phenotype in MSCs and highlights the potential of utilizing alginate hydrogels for MSC-based endochondral bone tissue engineering applications.

Engineering articular cartilage-like grafts by self-assembly of infrapatellar fat pad-derived stem cells.

Well documented limitations associated with primary chondrocytes for cartilage tissue engineering applications have led to increased interest in the use of multi-potent stem/progenitor cells. The objective of this study was to firstly investigate if infrapatellar fat pad-derived stem cells (FPSCs) could be used to engineer cartilage-like tissues through a self-assembly (SA) process, and secondly to compare the properties of such grafts to those engineered by agarose hydrogel encapsulation (AE). Self-assembled cartilaginous tissues were first engineered by geometrically confining FPSCs on tissue culture plastic, and then either continuously or transiently supplementing these constructs with transforming growth factor-b3 (TGF-b3). Transient supplementation with TGF-b3 (for the first 21 days of culture) enhanced the development of self-assembled grafts, with sGAG accumulation reaching levels of 8.4 ± 1.5% w/w after 6 weeks of culture. While overall levels of matrix synthesis were higher with AE compared to SA, when normalized to tissue wet weight, ECM accumulation was significantly greater in the lighter SA constructs. A potential drawback with the SA approach on tissue culture plastic was that it often led to the development of contracted,geometrically inconsistent tissues.We therefore next explored if SA on polyethylene terephthalate (PET) transwell membranes would lead to the development of more morphologically stable and homogenous tissues. At high seeding densities, SA on such transwell membranes led to the formation of geometrically uniform constructs that underwent minimal contraction during culture. In conclusion, the results of this study demonstrate the potential of SA using FPSCs for cartilage tissue engineering, with grafts attaining relatively high levels of sGAG content within clinically relevant timeframes. Such an approach is easily scalable and may lend itself to treating large, full thickness cartilage defects.

A comparison of self-assembly and hydrogel encapsulation as a means to engineer functional cartilaginous grafts using culture expanded chondrocytes.

Despite an increased interest in the use of hydrogel encapsulation and cellular self-assembly (often termed "self-aggregating" or "scaffold-free" approaches) for tissue-engineering applications, to the best of our knowledge, no study to date has been undertaken to directly compare both approaches for generating functional cartilaginous grafts. The objective of this study was to directly compare self-assembly (SA) and agarose hydrogel encapsulation (AE) as a means to engineer such grafts using passaged chondrocytes. Agarose hydrogels (5 mm diameter × 1.5 mm thick) were seeded with chondrocytes at two cell seeding densities (900,000 cells or 4 million cells in total per hydrogel), while SA constructs were generated by adding the same number of cells to custom-made molds. Constructs were either supplemented with transforming growth factor (TGF)-ß3 for 6 weeks, or only supplemented with TGF-ß3 for the first 2 weeks of the 6 week culture period. The SA method was only capable of generating geometrically uniform cartilaginous tissues at high seeding densities (4 million cells). At these high seeding densities, we observed that total sulphated glycosaminoglycan (sGAG) and collagen synthesis was greater with AE than SA, with higher sGAG retention also observed in AE constructs. When normalized to wet weight, however, SA constructs exhibited significantly higher levels of collagen accumulation compared with agarose hydrogels. Furthermore, it was possible to engineer such functionality into these tissues in a shorter timeframe using the SA approach compared with AE. Therefore, while large numbers of chondrocytes are required to engineer cartilaginous grafts using the SA approach, it would appear to lead to the faster generation of a more hyaline-like tissue, with a tissue architecture and a ratio of collagen to sGAG content more closely resembling native articular cartilage.

Scaffold architecture determines chondrocyte response to externally applied dynamic compression.

It remains unclear how specific mechanical signals generated by applied dynamic compression (DC) regulate chondrocyte biosynthetic activity. It has previously been suggested that DC-induced interstitial fluid flow positively impacts cartilage-specific matrix production. Modifying fluid flow within dynamically compressed hydrogels therefore represents a promising approach to controlling chondrocyte behavior, which could potentially be achieved by changing the construct architecture. The objective of this study was to first determine the influence of construct architecture on the mechanical environment within dynamically compressed agarose hydrogels using finite element (FE) modeling and to then investigate how chondrocytes would respond to this altered environment. To modify construct architecture, an array of channels was introduced into the hydrogels. Increased magnitudes of fluid flow were predicted in the periphery of dynamically compressed solid hydrogels and also around the channels in the dynamically compressed channeled hydrogels. DC was found to significantly increase sGAG synthesis in solid constructs, which could be attributed at least in part to an increase in DNA. DC was also found to preferentially increase collagen accumulation in regions of solid and channeled constructs where FE modeling predicted higher levels of fluid flow, suggesting that this stimulus is important for promoting collagen production by chondrocytes embedded in agarose gels. In conclusion, this study demonstrates how the architecture of cell-seeded scaffolds or hydrogels can be modified to alter the spatial levels of biophysical cues throughout the construct, leading to greater collagen accumulation throughout the engineered tissue rather than preferentially in the construct periphery. This system also provides a novel approach to investigate how chondrocytes respond to altered levels of biophysical stimulation.