Injectable In Situ–Forming pH=Thermo-Sensitive Hydrogel for Bone Tissue Engineering Hea Kyung Kim, M.S., 1 Woo Sun Shim, Ph.D., 1 Sung Eun Kim, Ph.D., 2 Kweon-Haeng Lee, M.D., Ph.D., 2 Eunah Kang, Ph.D., 3 Jong-Ho Kim, Ph.D., 3 Kwangmeyung Kim, Ph.D., 3 Ick Chan Kwon, Ph.D., 3 and Doo Sung Lee, Ph.D. 1 We developed a novel pH- and thermo-sensitive hydrogel as a scaffold for autologous bone tissue engineer- ing. We synthesized this polymer by adding pH-sensitive sulfamethazine oligomers (SMOs) to both ends of a thermo-sensitive poly(e-caprolactone-co-lactide)–poly(ethylene glycol)–poly(e-caprolactone-co-lactide) (PCLA- PEG-PCLA) block copolymer, yielding a pH=thermo-sensitive SMO-PCLA-PEG-PCLA-SMO block copolymer. The synthesized block copolymer solution rapidly formed a stable gel under physiological conditions (pH 7.4 and 378C), whereas it formed a sol at pH 8.0 and 378C, making it injectable. This pH=thermo-sensitive hydrogel exhibited high biocompatibility in a Dulbecco’s modified Eagle’s medium extract test. Under physiological conditions, the hydrogel easily encapsulated human mesenchymal stem cells (hMSCs) and recombinant human bone morphogenetic protein-2 (rhBMP-2), with encapsulating efficiencies of about 90% and 85%, respectively. To assay for ectopic bone formation in vivo, we subcutaneously injected a polymer solution containing hMSCs and rhBMP-2 into the back of mice, after which we could observe hMSC differentiation for up to 7 weeks. Histo- logical studies revealed mineralized tissue formation and high levels of alkaline phosphatase activity in the mineralized tissue. Therefore, this pH=thermo-sensitive SMO-PCLA-PEG-PCLA-SMO block copolymer dem- onstrated potential as an injectable scaffold for bone tissue engineering, with in situ formation capabilities. Introduction B one tissue engineering requires the interaction of a scaffold with osteocompetent cells and osteoinductive growth factors, in order to mediate cell differentiation and proliferation into the desired cell lineage. 1–3 To this end, many studies have investigated delivering mesenchymal stem cells (MSCs) or tissue-specific growth factors within natural or synthetic scaffolds, in order to induce autologous bone for- mation. 4–7 When MSCs communicate with tissue-specific growth factors, they act as pluripotent progenitor cells and can commit and differentiate into myoblasts, adipocytes, or osteoblasts, depending upon the specific conditions. 8–10 Members of the osteoinductive bone morphogenic protein (BMP) family are often used as stimulators to communicate with MSCs. BMPs initiate a signaling pathway by binding to a transmembrane serine-threonine kinase receptor on the MSC surface. 11 The resulting BMP signaling cascade induces com- mitment and terminal differentiation of MSCs, which ulti- mately leads to new bone formation. 12–14 In particular, several bone tissue engineering studies have co-delivered BMP-2 and bone marrow MSCs. 11,15,16 Various synthetic or natural scaffolds for MSCs and=or BMPs delivery have been described, including peptides, 17,18 collagen, 19 poly(fumarate), 20,21 methyl cellulose, 22 and poly (lactic-co-glycolic acid). 23,24 Hydrolysis of some biodegrad- able scaffolds can produce acidic side products that cause inflammation. 25 Since this acidic environment accelerates scaffold degradation, these scaffolds may also not retain the mechanical properties required for new bone formation. In addition, traditional scaffolds are pre-formed prior to graft- ing, whereas bone defects are often irregular in shape, re- quiring a free-forming scaffold that can be applied in a minimally invasive manner. In this regard, injectable scaffolds are now replacing traditional preformed solid scaffolds in bone tissue engineering. 26–28 Ideally, these scaffolds are liquid upon injection, foregoing an open surgical procedure, but they undergo gelation in situ, allowing them to fill in a bone defect of any shape. In addition, therapeutic agents such as growth factors and cytokines are more easily incorporated 1 Department of Polymer Science & Engineering, Sungkyunkwan University, Suwon, Republic of Korea. 2 Department of Pharmacology, The Catholic University of Korea, Seoul, Republic of Korea. 3 Biomedical Research Center, Korea Institute of Science and Technology, Seoul, Republic of Korea. TISSUE ENGINEERING: Part A Volume 15, Number 4, 2009 ª Mary Ann Liebert, Inc. DOI: 10.1089=ten.tea.2007.0407 923