Autologous bone grafting is the most effective treatment for long-bone nonunions, but it poses considerable risks to donors, necessitating the development of alternative therapeutics. Poly(ethylene glycol) (PEG) microencapsulation and BMP2 transgene delivery are being developed together to induce rapid bone formation. However, methods to make these treatments available for clinical applications are presently lacking. In this study we used mesenchymal stem cells (MSCs) due to their ease of harvest, replication potential, and immunomodulatory capabilities. MSCs were from sheep and pig due to their appeal as large animal models for bone nonunion. We demonstrated that cryopreservation of these microencapsulated MSCs did not affect their cell viability, adenoviral BMP2 production, or ability to initiate bone formation. Additionally, microspheres showed no appreciable damage from cryopreservation when examined with light and electron microscopy. These results validate the use of cryopreservation in preserving the viability and functionality of PEG-encapsulated BMP2-transduced MSCs. 1. Introduction Bone is the second most transplanted tissue behind blood transfusions [1] with 500,000 people in the US and 2.2 million people worldwide receiving bone grafts per year [2]. Autologous bone grafting is currently considered the gold standard for treating nonhealing fractures [3], but multiple features make it less than ideal for long bone nonunion treatment. The most promising graft donor site, the iliac crest, is available in limited quantities [4]. Since long bone nonunions can require up to 30？mLs of marrow, the amount harvested from the iliac crest can be insufficient [5]. Bone grafting presents considerable risks to patients by increased surgical times and blood loss [6], with 1/3 of patients experiencing chronic pain 24 months after transplant [7], and recipients are at increased risk for donor site instability and fractures [8]. Additionally, large bone defects, like those received by soldiers injured in combat [9, 10], often do not heal without surgical intervention and can end in an undesirable outcome such as amputation [11]. Bone morphogenetic protein 2 (BMP2) is a potential therapeutic that can fill the need for bone healing. Recombinant BMP2 can induce rapid ossification in orthopedic applications [12, 13] but has a relatively short half-life, must be administered at high dosages, and continually maintained to promote extensive and expedited bone regeneration [14–16]. Having a fast and maintained release/production of BMP2 as an off the shelf therapeutic
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