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Effective bone tissue engineering can restore bone and skeletal functions that are impaired by traumas and/or certain medical conditions. Bone is a complex tissue and functions through orchestrated interactions between cells, biomechanical forces, and biofactors. To identify ideal scaffold materials for effective mesenchymal stem cell (MSC)-based bone tissue regeneration, here we develop and characterize a composite nanoparticle hydrogel by combining carboxymethyl chitosan (CMCh) and amorphous calcium phosphate (ACP) (designated as CMCh-ACP hydrogel). We demonstrate that the CMCh-ACP hydrogel is readily prepared by incorporating glucono δ-lactone (GDL) into an aqueous dispersion or rehydrating the acidic freeze-dried nanoparticles in a pH-triggered controlled-assembly fashion. The CMCh-ACP hydrogel exhibits excellent biocompatibility and effectively supports MSC proliferation and cell adhesion. Moreover, while augmenting BMP9-induced osteogenic differentiation, the CMCh-ACP hydrogel itself is osteoinductive and induces the expression of osteoblastic regulators and bone markers in MSCs in vitro. The CMCh-ACP scaffold markedly enhances the efficiency and maturity of BMP9-induced bone formation in vivo, while suppressing bone resorption occurred in long-term ectopic osteogenesis. Thus, these results suggest that the pH-responsive self-assembled CMCh-ACP injectable and bioprintable hydrogel may be further exploited as a novel scaffold for osteoprogenitor-cell-based bone tissue regeneration.

Effective bone tissue engineering is important to overcome the unmet clinical challenges as more than 1.6 million bone grafts are done annually in the United States. Successful bone tissue engineering needs minimally three critical constituents: osteoprogenitor cells, osteogenic factors, and osteoinductive/osteoconductive scaffolds. Osteogenic progenitors are derived from multipotent mesenchymal stem cells (MSCs), which can be prepared from numerous tissue sources, including adipose tissue. We previously showed that BMP9 is the most osteogenic BMP and induces robust bone formation of immortalized mouse adipose-derived MSCs entrapped in a citrate-based thermoresponsive hydrogel referred to as PPCNg. As graphene and its derivatives emerge as promising biomaterials, here we develop a novel thermosensitive and injectable hybrid material by combining graphene oxide (GO) with PPCNg (designated as GO-P) and characterize its ability to promote bone formation. We demonstrate that the thermoresponsive behavior of the hybrid material is maintained while effectively supporting MSC survival and proliferation. Furthermore, GO-P induces early bone-forming marker alkaline phosphatase (ALP) and potentiates BMP9-induced expression of osteogenic regulators and bone markers as well as angiogenic factor VEGF in MSCs. In vivo studies show BMP9-transduced MSCs entrapped in the GO-P scaffold form well-mineralized and highly vascularized trabecular bone. Thus, these results indicate that GO-P hybrid material may function as a new biocompatible, injectable scaffold with osteoinductive and osteoconductive activities for bone regeneration.

Graphene-based materials are used in many fields but have found only limited applications in biomedicine, including bone tissue engineering. Here, we demonstrate that novel hybrid materials consisting of gelatin-derived graphene and silicate nanosheets of Laponite (GL) are biocompatible and promote osteogenic differentiation of mesenchymal stem cells (MSCs). Homogeneous cell attachment, long-term proliferation, and osteogenic differentiation of MSCs on a GL-scaffold were confirmed using optical microscopy and scanning electron microscopy. GL-powders made by pulverizing the GL-scaffold were shown to promote bone morphogenetic protein (BMP9)-induced osteogenic differentiation. GL-powders increased the alkaline phosphatase (ALP) activity in immortalized mouse embryonic fibroblasts but decreased the ALP activity in more-differentiated immortalized mouse adipose-derived cells. Note, however, that GL-powders promoted BMP9-induced calcium mineral deposits in both MSC lines, as assessed using qualitative and quantitative alizarin red assays. Furthermore, the expression of chondro-osteogenic regulator markers such as Runx2, Sox9, osteopontin, and osteocalcin was upregulated by the GL-powder, independent of BMP9 stimulation; although the powder synergistically upregulated the BMP9-induced Osterix expression, the adipogenic marker PPARγ was unaffected. Furthermore, in vivo stem cell implantation experiments demonstrated that GL-powder could significantly enhance the BMP9-induced ectopic bone formation from MSCs. Collectively, our results strongly suggest that the GL hybrid materials promote BMP9-induced osteogenic differentiation of MSCs and hold promise for the development of bone tissue engineering platforms.