Freshly isolated stromal cells can potentially be used as an alternative to in vitro expanded cells in regenerative medicine. Their use requires the development of bioactive hydrogels or scaffolds which provide an environment to enhance their proliferation and tissue-specific differentiation in vivo. The goal of the current study was to develop an injectable fibrin hydrogel functionalized with cartilage ECM microparticles and transforming growth factor (TGF)-β3 as a putative therapeutic for articular cartilage regeneration. ECM microparticles were produced by cryomilling and freeze-drying porcine articular cartilage. Up to 2% (w/v) ECM could be incorporated into fibrin without detrimentally affecting its capacity to form stable hydrogels. To access the chondroinductivity of cartilage ECM, we compared chondrogenesis of infrapatellar fat pad-derived stem cells in fibrin hydrogels functionalized with either particulated ECM or control gelatin microspheres. Cartilage ECM particles could be used to control the delivery of TGF-β3 to IFP-derived stem cells within fibrin hydrogels in vitro, and furthermore, led to higher levels of sulphated glycosaminoglycan (sGAG) and collagen accumulation compared to control constructs loaded with gelatin microspheres. In vivo, freshly isolated stromal cells generated a more cartilage-like tissue within fibrin hydrogels functionalized with cartilage ECM particles compared to the control gelatin loaded constructs. These tissues stained strongly for type II collagen and contained higher levels of sGAGs. These results support the use of fibrin hydrogels functionalized with cartilage ECM components in single-stage, cell-based therapies for joint regeneration. An alternative to the use of in vitro expanded cells in regenerative medicine is the use of freshly isolated stromal cells, where a bioactive scaffold or hydrogel is used to provide an environment that enhances their proliferation and tissue-specific differentiation in vivo. The objective of this study was to develop an injectable fibrin hydrogel functionalized with cartilage ECM micro-particles and the growth factor TGF-β3 as a therapeutic for articular cartilage regeneration. This study demonstrates that freshly isolated stromal cells generate cartilage tissue in vivo when incorporated into such a fibrin hydrogels functionalized with cartilage ECM particles. These findings open up new possibilities for in-theatre, single-stage, cell-based therapies for joint regeneration.

Joint-derived stem cells are a promising alternative cell source for cartilage repair therapies that may overcome many of the problems associated with the use of primary chondrocytes (CCs). The objective of this study was to compare the in vitro functionality and in vivo phenotypic stability of cartilaginous tissues engineered using bone marrow-derived stem cells (BMSCs) and joint tissue-derived stem cells following encapsulation in agarose hydrogels. Culture-expanded BMSCs, fat pad-derived stem cells (FPSCs), and synovial membrane-derived stem cells (SDSCs) were encapsulated in agarose and maintained in a chondrogenic medium supplemented with transforming growth factor-β3. After 21 days of culture, constructs were either implanted subcutaneously into the back of nude mice for an additional 28 days or maintained for a similar period in vitro in either chondrogenic or hypertrophic media formulations. After 49 days of in vitro culture in chondrogenic media, SDSC constructs accumulated the highest levels of sulfated glycosaminoglycan (sGAG) (∼2.8% w/w) and collagen (∼1.8% w/w) and were mechanically stiffer than constructs engineered using other cell types. After subcutaneous implantation in nude mice, sGAG content significantly decreased for all stem cell-seeded constructs, while no significant change was observed in the control constructs engineered using primary CCs, indicating that the in vitro chondrocyte-like phenotype generated in all stem cell-seeded agarose constructs was transient. FPSCs and SDSCs appeared to undergo fibrous dedifferentiation or resorption, as evident from increased collagen type I staining and a dramatic loss in sGAG content. BMSCs followed a more endochondral pathway with increased type X collagen expression and mineralization of the engineered tissue. In conclusion, while joint tissue-derived stem cells possess a strong intrinsic chondrogenic capacity, further studies are needed to identify the factors that will lead to the generation of a more stable chondrogenic phenotype.

Agarose hydrogels are commonly used for cartilage tissue engineering studies and to provide a three dimensional environment to investigate cellular mechanobiology. Interpreting the results of such studies requires accurate quantification of the mechanical properties of the hydrogel. There is significant variation in the reported mechanical properties of agarose hydrogels, and little is reported on the influence of factors associated with mixing these hydrogels with cell suspensions on their initial mechanical properties. The objective of this study was to determine the influence of agarose concentration, the cooling rate during gelation, the thermal history following gelation and the cell seeding density on the initial mechanical properties of agarose hydrogels. The average ramp modulus of 2% agarose gel in tension was 24.9 kPa (+/-1.7, n=10), compared with 55.6 kPa (+/-0.5, n=10) in compression. The average tensile equilibrium modulus was 39.7 kPa (+/-5.7, n=6), significantly higher than the compressive equilibrium modulus of 14.2 kPa (+/-1.6, n=10). The equilibrium and dynamic compressive modulus of agarose hydrogels were observed to reduce if maintained at 37 ( composite function)C following gelation compared with samples maintained at room temperature. Depending on the methodology used to encapsulate chondrocytes within agarose hydrogels, the equilibrium compressive modulus was found to be significantly higher for acellular 2% agarose gels compared with 2% agarose gels seeded at approximately 40 x 10(6) cells/mL. These results have important implications for interpreting the results of chondrocyte mechanobiology studies in agarose hydrogels.

Chondrogenically primed bone marrow-derived mesenchymal stem cells (MSCs) have been shown to become hypertrophic and undergo endochondral ossification when implanted in vivo. Modulating this endochondral phenotype may be an attractive approach to engineering the osseous phase of an osteochondral implant. The objective of this study was to engineer an osteochondral tissue by promoting endochondral ossification in one layer of a bilayered construct and stable cartilage in the other. The top half of bilayered agarose hydrogels were seeded with culture expanded chondrocytes (termed the chondral layer) and the bottom half of the bilayered agarose hydrogels with MSCs (termed the osseous layer). Constructs were cultured in chondrogenic medium for 21days and thereafter were either maintained in chondrogenic medium, transferred to hypertrophic medium, or implanted subcutaneously into nude mice. This structured chondrogenic bilayered co-culture was found to enhance chondrogenesis in the chondral layer, appearing to help re-establish the chondrogenic phenotype that is lost in chondrocytes during monolayer expansion. Furthermore, the bilayered co-culture appeared to suppress hypertrophy and mineralization in the osseous layer. The addition of hypertrophic factors to the media was found to induce mineralization of the osseous layer in vitro. A similar result was observed in vivo where endochondral ossification was restricted to the osseous layer of the construct, leading to the development of an osteochondral tissue. This novel approach represents a potential new treatment strategy for the repair of osteochondral defects.