Biomaterials-based approaches to harnessing the immune and inflammatory responses to potentiate wound healing hold important promise. Bone fracture healing is characterized by an acute inflammatory phase, followed by a transition to a regenerative and repair phase. In this study, we developed genipin-crosslinked gelatin microspheres designed to be preferentially degraded by inflammatory (M1) macrophages. Highly crosslinked (>90%) microspheres allowed efficient incorporation of bioactive bone morphogenetic protein 2 (BMP2), a potent stimulator of osteogenesis in progenitor cells, via electrostatic interactions. Release of BMP2 was directly correlated with degradation of the gelatin matrix. Exposure of microspheres to polarized murine macrophages showed that degradation was significantly higher in the presence of M1 macrophages, relative to alternatively activated (M2) macrophages and unpolarized controls. Microsphere degradation in the presence of non-inflammatory cells resulted in very low degradation rates. The expression of matrix metalloproteinases (MMPs) and tissue inhibitors of MMP (TIMPs) by macrophages were consistent with the observed phenotype-dependent degradation rates. Indirect co-culture of BMP2-loaded microspheres and macrophages with isolated adipose-derived mesenchymal stem cells (MSC) showed that M1 macrophages produced the strongest osteogenic response, comparable to direct supplementation of the culture medium with BMP2. Controlled release systems that are synchronized with the inflammatory response have the potential to provide better spatiotemporal control of growth factor delivery and therefore may improve the outcomes of recalcitrant wounds.

Repair of complex fractures with bone loss requires a potent, space-filling intervention to promote regeneration of bone. We present a biomaterials-based strategy combining mesenchymal stromal cells (MSC) with a chitosan-collagen matrix to form modular microtissues designed for delivery through a needle to conformally fill cavital defects. Implantation of microtissues into a calvarial defect in the mouse showed that osteogenically pre-differentiated MSC resulted in complete bridging of the cavity, while undifferentiated MSC produced mineralized tissue only in apposition to native bone. Decreasing the implant volume reduced bone regeneration, while increasing the MSC concentration also attenuated bone formation, suggesting that the cell-matrix ratio is important in achieving a robust response. Conformal filling of the defect with microtissues in a carrier gel resulted in complete healing. Taken together, these results show that modular microtissues can be used to augment the differentiated function of MSC and provide an extracellular environment that potentiates bone repair.

A modular tissue engineering approach may have advantages over current therapies in providing rapid and sustained revascularization of ischemic tissue. In this study, modular protein microbeads were prepared from pure fibrin (FIB) and collagen-fibrin composites (COL-FIB) using a simple water-in-oil emulsification technique. Human endothelial cells and fibroblasts were embedded directly in the microbead matrix. The resulting microbeads were generally spheroidal with a diameter of 100–200 μm. Cell viability was high (75–80% viable) in microbeads, but was marginally lower than in bulk hydrogels of corresponding composition (85–90% viable). Cell proliferation was significantly greater in COL-FIB microbeads after two weeks in culture, compared to pure FIB microbeads. Upon embedding of microbeads in a surrounding fibrin hydrogel, endothelial cell networks formed inside the microbead matrix and extended into the surrounding matrix. The number of vessel segments, average segment length, and number of branch points was higher in FIB samples, compared to COL-FIB samples, resulting in significantly longer total vessel networks. Anastomosis of vessel networks from adjacent microbeads was also observed. These studies demonstrate that primitive vessel networks can be formed by modular protein microbeads containing embedded endothelial cells and fibroblasts. Such microbeads may find utility as prevascularized tissue modules that can be delivered minimally invasively as a therapy to restore blood flow to ischemic tissues.

Despite innovations in surgical interventions, treatment of cartilage injury in osteoarthritic joints remains a challenge due to concomitant inflammation. Obstructing a single dominant inflammatory cytokine has shown remarkable clinical benefits in rheumatoid arthritis, and similar strategies are being suggested to target inflammatory pathways in osteoarthritis (OA). Here, we describe the utility of gelatin microspheres that are responsive to proteolytic enzymes typically expressed in arthritic flares, resulting in on-demand and spatiotemporally controlled release of antiinflammatory cytokines for cartilage preservation and repair. These microspheres were designed with a net negative charge to sequester cationic anti-inflammatory cytokines, and the magnitude of the negative charge potential increased with an increase in crosslinking density. Collagenase-mediated degradation of the microspheres was dependent on the concentration of the enzyme. Release of antiinflammatory cytokines from the loaded microspheres directly correlated with the degradation of the gelatin matrix. Exposure of the IL-4 and IL-13 loaded microspheres reduced the inflammation of chondrocytes up to 80%. Hence, the delivery of these microspheres in an OA joint can attenuate the stimulation of chondrocytes and the resulting secretion of catabolic factors such as proteinases and nitric oxide. The microsphere format also allows for minimally invasive delivery and is less susceptible to mechanically induced drug release. Consequently, bioresponsive microspheres can be an effective tool for cartilage preservation and arthritis treatment.

Background Cell-based therapies have made an impact on the treatment of osteoarthritis, however the repair and regeneration of thick cartilage defects is an important and growing clinical problem. Next-generation therapies that combine cells with biomaterials may provide improved outcomes. We have developed modular microenvironments that mimic the composition of articular cartilage as a delivery system for consistently differentiated cells. Methods Human bone marrow-derived mesenchymal stem cells (MSC) were embedded in modular microbeads consisting of agarose (AG) supplemented with 0%, 10%, and 20% collagen Type II (COL-II) using a water-in-oil emulsion technique. AG and AG/COL-II microbeads were characterized in terms of their structural integrity, size distribution, and protein content. The viability of embedded MSC and their ability to differentiate into osteogenic, adipogenic and chondrogenic lineages over three weeks in culture were also assessed. Results Microbeads made with <20% COL-II were robust, generally spheroidal in shape and 80 ± 10 µm in diameter. MSC viability in microbeads was consistently high over a week in culture, while viability in corresponding bulk hydrogels decreased with increasing COL-II content. Osteogenic differentiation of MSC was modestly supported in both AG and AG/COL-II microbeads, while adipogenic differentiation was strongly inhibited in COL-II containing microbeads. Chondrogenic differentiation of MSC was clearly promoted in microbeads containing COL-II, compared to pure AG matrices. Conclusions Inclusion of collagen Type II in agarose matrices in microbead format can potentiate chondrogenic differentiation of human MSC. Such compositionally tailored microtissues may find utility for cell delivery in next-generation cartilage repair therapies.