Statistics. More information about statistics is available in the Supplemental Methods. Study approval. All animal procedures were carried out in accordance with the guidelines provided in the Guide for the Use and Care of Laboratory Animals (National Academies Press, 2011) and were approved by the IACUC of the University of Michigan (PRO0007930).

Bone repair requires the tightly regulated control of multiple intrinsic and extrinsic cell types and signaling pathways. One of the positive regulatory signaling pathways in membranous and endochondral bone healing is the Hedgehog (Hh) signaling family. Here, a novel therapeutic liposomal delivery vector was developed by self-assembly of an Hh-activating cholesterol analog with an emulsifier, along with the addition of Smoothened agonist (SAG) as a drug cargo, for the enhancement of Hh signaling in bone regeneration. The drug-loaded nanoparticulate agonists of Hh signaling were immobilized onto trabecular bone-mimetic apatite-coated 3D scaffolds using bioinspired polydopamine adhesives to ensure favorable microenvironments for cell growth and local therapeutic delivery. Results showed that SAG-loaded liposomes induced a significant and dose-dependent increase in Hh-mediated osteogenic differentiation, as evidenced by in vitro analysis of bone marrow stromal cells, and in vivo calvarial bone healing, as evidenced using all radiographic parameters and histomorphometric analyses. Moreover, favorable outcomes were achieved in comparison to standards of care, including collagen sponge-delivered rBMP2 or allograft bone. In summary, this study demonstrates using a nanoparticle packaged Hh small molecule as a widely applicable bone graft substitute for robust bone repair.

The vascular wall stores mesenchymal progenitor cells which are able to induce bone regeneration, via direct and paracrine mechanisms. Although much is known regarding perivascular cell regulation of osteoblasts, their regulation of osteoclasts, and by extension utility in states of high bone resorption, is not known. Here, human perivascular stem cells (PSCs) were used as a means to prevent autograft resorption in a gonadectomy‐induced osteoporotic spine fusion model. Furthermore, the paracrine regulation by PSCs of osteoclast formation was evaluated, using coculture, conditioned medium, and purified extracellular vesicles. Results showed that PSCs when mixed with autograft bone induce an increase in osteoblast:osteoclast ratio, promote bone matrix formation, and prevent bone graft resorption. The confluence of these factors resulted in high rates of fusion in an ovariectomized rat lumbar spine fusion model. Application of PSCs was superior across metrics to either the use of unpurified, culture‐defined adipose‐derived stromal cells or autograft bone alone. Under coculture conditions, PSCs negatively regulated osteoclast formation and did so via secreted, nonvesicular paracrine factors. Total RNA sequencing identified secreted factors overexpressed by PSCs which may explain their negative regulation of graft resorption. In summary, PSCs reduce osteoclast formation and prevent bone graft resorption in high turnover states such as gonadectomy‐induced osteoporosis.

Progenitor cells from adipose tissue are able to induce bone repair; however, inconsistent or unreliable efficacy has been reported across preclinical and clinical studies. Soluble inhibitory factors, such as the secreted Wnt signaling antagonists Dickkopf-1 (DKK1), are expressed to variable degrees in human adipose-derived stem cells (ASCs), and may represent a targetable "molecular brake" on ASC mediated bone repair. Here, anti-DKK1 neutralizing antibodies were observed to increase the osteogenic differentiation of human ASCs in vitro, accompanied by increased canonical Wnt signaling. Human ASCs were next engrafted into a femoral segmental bone defect in NOD-Scid mice, with animals subsequently treated with systemic anti-DKK1 or isotype control during the repair process. Human ASCs alone induced significant but modest bone repair. However, systemic anti-DKK1 induced an increase in human ASC engraftment and survival, an increase in vascular ingrowth, and ultimately improved bone repair outcomes. In summary, anti-DKK1 can be used as a method to augment cell-mediated bone regeneration, and could be particularly valuable in the contexts of impaired bone healing such as osteoporotic bone repair. K E Y W O R D S adipose stem cell, adipose stromal cell, bone healing, bone repair, bone tissue engineering, mesenchymal stem cell, Wnt signaling 1 | INTRODUCTION Nonhealing skeletal defects are addressed in millions of surgeries worldwide each year, in diverse fields such as orthopedic, neurocranial, plastic, and oral and dental surgery. Segmental bone defects can be the result of congenital abnormalities, or can arise secondarily from diverse causes such as trauma, malignancy, or infection. In adult patients, a critical defect of long bones is defined as a bone loss involving >50% of the circumference or >2 cm in length. 1 The available techniques to surgical manage these conditions are mainly based on bone grafting, distraction osteogenesis, and the induced membrane technique. 2 These approaches require long times of recovery and Stefano Negri and Yiyun Wang contributed equally to this work.