Self-assembling cell sheets have shown great potential for use in cartilage tissue engineering applications, as they provide an advantageous environment for the chondrogenic induction of human mesenchymal stem cells (hMSCs). We have engineered a system of self-assembled, microsphere-incorporated hMSC sheets capable of forming cartilage in the presence of exogenous transforming growth factor β1 (TGF-β1) or with TGF-β1 released from incorporated microspheres. Gelatin microspheres with two different degrees of crosslinking were used to enable different cell-mediated microsphere degradation rates. Biochemical assays, histological and immunohistochemical analyses, and biomechanical testing were performed to determine biochemical composition, structure, and equilibrium modulus in unconfined compression after 3 weeks of culture. The inclusion of microspheres with or without loaded TGF-β1 significantly increased sheet thickness and compressive equilibrium modulus, and enabled more uniform matrix deposition by comparison to control sheets without microspheres. Sheets incorporated with fast-degrading microspheres containing TGF-β1 produced significantly more GAG and GAG per DNA than all other groups tested and stained more intensely for type II collagen. These findings demonstrate improved cartilage formation in microsphere-incorporated cell sheets, and describe a tailorable system for the chondrogenic induction of hMSCs without necessitating culture in growth factor-containing medium.

There is an increasing need to control the type, quantity, and timing of growth factors released during tissue healing. Sophisticated delivery systems offering the ability to deliver multiple growth factors with independently tunable kinetics are highly desirable. Here, a multilayered, mineral coated micro-particle (MCMs) platform that can serve as an adaptable dual growth factor delivery system is developed. Bone morphogenetic protein-2 (BMP-2) and vascular endothelial growth factor (VEGF) are bound to the mineral coatings with high binding efficiencies of up to 80%. BMP-2 is firstly bound onto a 1st mineral coating layer; then VEGF is bound onto a 2nd mineral coating layer. The release of BMP-2 is sustained over a period of 50 days while the release of VEGF is a typical two-phase release with rapid release in the first 14 days and more sustained release for the following 36 days. Notably, the release behaviors of both growth factors can be independently tailored by changing the intrinsic properties of the mineral coatings. Furthermore, the release of BMP-2 can be tuned by changing the thickness of the 2nd layer. This injectable microparticle based delivery platform with tunable growth factor release has immense potential for applications in tissue engineering and regenerative medicine.

A microparticle-based growth factor delivery system was engineered to drive endochondral ossification within human bone marrow-derived mesenchymal stem cell (hMSC) aggregates. Compared with cell-only aggregates treated with exogenous growth factors, aggregates with incorporated transforming growth factor-β1- and BMP-2-loaded microparticles exhibited enhanced chondrogenesis and alkaline phosphatase activity and a greater degree of mineralization. This microparticle-incorporated system has potential as a readily implantable therapy for healing bone defects without the need for long-term in vitro chondrogenic priming.

The precise spatial and temporal presentation of growth factors is critical for cartilage development, during which tightly controlled patterns of signals direct cell behavior and differentiation. Recently, chondrogenic culture of human mesenchymal stem cells (hMSCs) has been improved through the addition of polymer microspheres capable of releasing growth factors directly to cells within cellular aggregates, eliminating the need for culture in transforming growth factor-β1 (TGF-β1)-containing medium. However, the influence of specific patterns of spatiotemporal growth factor presentation on chondrogenesis within microsphere-incorporated cell systems is unclear. In this study, we examined the effects of altering the chondrogenic microenvironment within hMSC aggregates through varying microsphere amount, growth factor concentration per microsphere, and polymer degradation time. Cartilage formation was evaluated in terms of DNA, glycosaminoglycan, and type II collagen in hMSCs from three donors. Chondrogenesis equivalent to or greater than that of aggregates cultured in medium containing TGF-β1 was achieved in some conditions, with varied differentiation based on the specific conditions of microsphere incorporation. A more spatially distributed delivery of TGF-β1 from a larger mass of fast-degrading microspheres improved differentiation by comparison with delivery from a smaller mass of microspheres with a higher TGF-β1 concentration per microsphere, although the total amount of growth factor per aggregate was the same. Results also indicated that the rate and degree of chondrogenesis varied on a donor-to-donor basis. Overall, this study elucidates the effects of varied conditions of TGF-β1-loaded microsphere incorporation on hMSC chondrogenesis, demonstrating that both spatiotemporal growth factor presentation and donor variability influence chondrogenic differentiation within microsphere-incorporated cellular constructs.

The growing socioeconomic burden of musculoskeletal injuries and limitations of current therapies have motivated tissue engineering approaches to generate functional tissues to aid in defect healing. A readily implantable scaffold‐free system comprised of human bone marrow‐derived mesenchymal stem cells embedded with bioactive microparticles capable of controlled delivery of transforming growth factor‐beta 1 (TGF‐β1) and bone morphogenetic protein‐2 (BMP‐2) was engineered to guide endochondral bone formation. The microparticles were formulated to release TGF‐β1 early to induce cartilage formation and BMP‐2 in a more sustained manner to promote remodeling into bone. Cell constructs containing microparticles, empty or loaded with one or both growth factors, were implanted into rat critical‐sized calvarial defects. Micro‐computed tomography and histological analyses after 4 weeks showed that microparticle‐incorporated constructs with or without growth factor promoted greater bone formation compared to sham controls, with the greatest degree of healing with bony bridging resulting from constructs loaded with BMP‐2 and TGF‐β1. Importantly, bone volume fraction increased significantly from 4 to 8 weeks in defects treated with both growth factors. Immunohistochemistry revealed the presence of types I, II, and X collagen, suggesting defect healing via endochondral ossification in all experimental groups. The presence of vascularized red bone marrow provided strong evidence for the ability of these constructs to stimulate angiogenesis. This system has great translational potential as a readily implantable combination therapy that can initiate and accelerate endochondral ossification in vivo. Importantly, construct implantation does not require prior lengthy in vitro culture for chondrogenic cell priming with growth factors that is necessary for current scaffold‐free combination therapies. Stem Cells Translational Medicine 2017;6:1644–1659