Wei Yao, M.D.
Principle Investigator – MSC on OP
Co-PI – CIRM project on OP
Co-Investigator – 3D Research Project
Project Leader: SCOR Project 1
Associate Professor
Associate Director – Laboratory Core
Center for Musculoskeletal Health
UC Davis
wei.yao@ucdmc.ucdavis.edu

Evan E. Lay, M.S.
Staff Research Associate, II
Center for Musculoskeletal Health
UC Davis
yaelay@ucdavis.edu

Kit S. Lam, M.D., Ph.D.
Professor and Chair, Department of Biochemistry and Molecular Medicine
Professor of Medicine
Division of Hematology & Oncology
UC Davis Cancer Center
UC Davis

Nancy E. Lane, M.D.
Co-Investigator - MSC on OP
Director - SCOR
Co-Project Leader - SCOR Project 1
Project Leader - SCOR Project 3
Endowed Professor of Medicine and Rheumatology
Director: Musculoskeletal Diseases of Aging Research Group
Co-Director: Building Interdisciplinary Research Careers in Women's Health (BIRCWH)
Co-Director: Center for Translational Research in Osteoarthritis
UC Davis
nancy.lane@ucdmc.ucdavis.edu

Ralph Marcucio, Ph.D.
Consultant – MSC on OP
Director, Laboratory for Skeletal Regeneration
Orthopedic Trauma Institute
Associate Professor
Department of Orthopaedic Surgery
UC San Francisco

Jan A. Nolta, Ph.D.
Director, Stem Cell Program and Institute for Regenerative Cures
Scientific Director, UC Davis GMP facility
Professor
UC Davis
Jan.nolta@ucdmc.ucdavis.edu

Robert O. Ritchie, Ph.D., Sc.D.
Consultant – MSC on OP
H.T. & Jessie Chua Distinguished Professor of Engineering
Professor and Chair, Department of Materials Science & Engineering
Professor of Mechanical Engineering, UC Berkeley
Senior Faculty Scientist, Materials Sciences Division, Lawrence Berkeley National Laboratory
ritchie@berkeley.edu

BACKGROUND

Aging reduces the number of mesenchymal stem cells (MSCs) in the bone marrow which leads to impairment of osteogenesis and subsequent bone loss (osteoporosis). Previous attempts to use systematic MSC transplantation have failed to regenerate bone due to inability of the transplanted MSCs to home to bone. MSCs transplantation may be a therapeutic option to treat bone loss if the cells could be directed to bone and induced to differentiate into osteoblasts. Our research group has developed a novel method to direct transplanted MSCs to bone by creating a compound with high affinity to both the α4β1 integrin on MSCs (a synthetic peptidomimetic ligand, LLP2A) and bone (alendronate). The resulting conjugate, LLP2A-Ale, significantly increased homing of the transplanted MSCs to bone and underwent osteoblast differentiation in a xenotransplantation model. LLP2A-Ale also increased bone formation and bone mass in young immune competent mice. We have shown that MSCs attach to bone and undergo osteogenic differentiation when they are “directed” to the bone surface. Although LLP2A-Ale is effective in young animals, the compound failed to significantly augment bone formation in aged animals unless it was used in combination with systemic MSC transplantation. Moreover, data on disease model is lacking. Therefore, we propose testing the effectiveness of LLP2A-Ale in directing the transplanted MSCs in augmenting bone formation in animal models of osteoporosis and fracture healing. We hypothesize that LLP2A-Ale will direct the transplanted MSCs to the bone and facilitate new bone formation in the treatments of severe osteoporosis caused by aging, hormone deficiency or other disorders.

SPECIFIC AIMS

Specific Aim #1. To determine the efficacy of LLP2A-Ale alone, or in combination with MSC transplantation, in the augmentation of bone formation in animal models of osteoporosis. We will evaluate if LLP2A-Ale alone, or in combination with transplanted MSCs will direct the donor MSCs to bone and augment bone formation to prevent or treat bone loss induced by estrogen-deficiency or with advanced aging. Endpoint measures will include ex vivo tracking of both the endogenous and the transplanted MSCs, osteoblast surface and numbers, bone formation, 3-dimensional bone architecture, serum biochemical and surface-based bone turnover and bone strength changes. The effects of LLP2A-Ale will be compared to an anti-resorptive agent, alendronate, and an anabolic agent, human PTH (1-34), a bone-growth agent.

Specific Aim #2. To determine if LLP2A-Ale alone, or in combination with MSCs will increase MSC engraftment and accelerate fracture healing. We will evaluate 1), how LLP2A-Ale affects MSC engraftment at the fracture site, time-dependent distribution,

and how it participates in chondrogenesis and osteogenesis and 2), how LLP2A-Ale and MSC transplantation affects the fracture healing process.

SIGNIFICANCE

Osteoporosis is a disease of increased bone fragility that results from estrogen deficiency and aging. It is a major public health problem with nearly 50% of all Caucasian women and 25% of Caucasian men at risk for an osteoporotic fracture in their lifetime (Publication from National Osteoporosis Foundation). With the aging segment of our population growing rapidly worldwide, osteoporosis has become a significant health concern.

Aging is associated with a reduction in marrow mesenchymal stem cell (MSC) numbers and a deficiency in the supportive mechanisms that involves them in the bone formation process [1-3]. The decrease in the resident MSC population with advanced age may be the most important factor responsible for reduced bone formation and the subsequent increase in bone fragility [4, 5]. Currently, nearly all of the treatments for osteoporosis reduce bone loss by decreasing osteoclastic bone resorption and thereby preventing further breakdown of bone. Importantly, this class of drugs does not restore the lost bone structure. Therapeutic modalities that target bone formation by either increasing the number of and/or activity of osteoblasts may be a more attractive approach to enhance bone formation and promote bone regeneration. Bone regeneration through induction of MSCs could promote osteogenesis and provide a rational therapeutic strategy for preventing age-related osteoporosis. Both autologous and allogeneic stem cells have been successfully infused for the treatments of degenerative heart, neuronal diseases or for injury repair [6-12]. However, systemic infusions of MSCs in vivo have failed to promote an osteogenic response in bone due to the inability of MSCs to home to the bone surface unless they were genetically modified [13-16] or following certain conditions such as injuries [17, 18]. This has become a major obstacle for MSC transplantation [19, 20]. Even if the transplanted MSCs make it to “bone”, they are usually observed engrafting in the upper metaphysis, epiphysis, within bone marrow in the sinusoids or within the haversian system [20-23]. The cells are removed from bone marrow by 4-8 weeks and do not show long term engraftment to bone tissue [20-22]. Thus far, no convincing data has demonstrated transplanted of MSCs that can home to bone and increase bone formation.

Mesenchymal stem cells within the bone marrow have multi-lineage potential and represent a mixture of precursors for mesenchymal - derived cell types including osteoblasts, chondrocytes and adipocytes [24- 26]. Bone marrow is the site where the committed osteoblast progenitors reside [16, 27]. The rigidity of the matrix where the MSCs reside could significantly influence the stem cells cytoskeletal contractility and differentiation [28]. Mobilization of the osteoblastic progenitors to the bone surface is a critical step that favors MSC osteoblast differentiation [28] to form mineralized tissue [29, 30]. Bone cells at all maturation stages are dependent on cell-matrix and cell-cell interactions [31-34]. Once the osteoblastic progenitors are “directed” to the bone surface, they synthesize a range of proteins including osteocalcin, osteopontin, bone sialoprotein, osteonectin, collagen-I and fibronectin that further enhance the adhesion and maturation of osteoblasts [35- 37]. These interactions are largely mediated by transmembrane integrin receptors that primarily utilize an arginine-glycine-aspartate (RGD) sequence to identify and bind to specific ligands. MSCs express integrins α1, 2, 3, 4, 6, 11, CD51 (integrin αV), and CD29 (integrins β1) [38]. Integrins α1β1, α2β1, αvβ1, αvβ5, α5β1and α4β1 are expressed in the osteoblastic cells [32, 36, 37, 39]. Integrin α5 is required for MSC osteogenic differentiation [40] and overexpression of α4 Integrin on MSCs has been reported to increase homing of the MSCs to bone [31]. These studies reveal that targeting the integrins on MSCs may facilitate bone regeneration.

We have previously reported the use of the one-bead-one compound (OBOC) combinatorial library method [24] to develop a high affinity and highly specific peptidomimetic ligand, LLP2A, against activated α4β1 integrin (IC50 = 2 pM) [41]. LLP2A, when conjugated to near infrared fluorescent dye, can be used to image α4β1-expressing cells in vivo with high sensitivity and specificity [41]. LLP2A partially reversed the airway inflammatory response in ovalbumin-induced inflammatory airway model in mice [42]. Scramble LLP2A ligand, however, loses its affinity to α4β1[41]. Collectively, the affinity of LLP2A-Ale for α4β1 integrin on MSCs formed the basis for our experiments [41, 43-47]. Bisphosphonates are widely used for the treatment of osteoporosis. This class of drugs is also used as “vehicle” for delivering bone-targeted drugs to osseous tissue as pro-drugs based on their biphosphonic moiety [48-50].

Bisphosphonates have been used to deliver sustained-release diclofenac, a non-steroidal anti-inflammatory drug to bone in rats [51]. The dose of bisphosphonate needed for this drug-delivery purpose is usually 10 to 100 fold lower than the doses needed for treatment of osteoporosis, hypocalcaemia, Paget’s disease or metastatic bone cancer. With this principle in mind, we have successfully conjugated the LLP2A to a bisphosphonate, alendronate, which serves as a bone seeking component, to “direct” LLP2A and its targeting MSCs to the bone surface. Our compound is designed to target both “bone” and “MSCs”. This approach for tissue regeneration could be useful for children in whom current osteoporosis medications are contraindicated, individuals who have had radiation to their skeletons, and for augmenting fracture healing.

Guan M1, Yao W, Liu R, Lam KS, Nolta J, Jia J, Panganiban B, Meng L, Zhou P, Shahnazari M, Ritchie RO, Lane NE. Directing mesenchymal stem cells to bone to augment bone formation and increase bone mass. A Bone: Homing MSCs to the surface of bones. [Nat Rev Rheumatol. 2012], Nature Medicine, 18; 486-462, 2012 PMID: 22306732 PMCID:PMC3755884 DOI: 10.1038/nm.2665.

Yao W, Guan M, Jia JJ, L Ya YA, Liu RW, Lam Kit, Nolta J, Olvera D, Ritchie RO, Lane NE. Reversal of bone loss induced by estrogen deficiency or aging by guiding the mesenchymal stem cells to bone.  Stem Cell 31:2004-2013, 2013.

Yao W, Lane NE, Targeted delivery of mesenchymal stem cells to the bone. Copyright © 2014 Elsevier Inc. All rights reserved. PMID:25173607 PMCID:PMC4268265 DOI: 10.1016/j.bone.2014.07.026

W. Yao, W. DaiL. JiangE. Y.-A. LayZ. ZhongR. O. RitchieX. LiH. KeN. E. Lane Slerostin-antibody treatment of glucocorticoid-induced osteoporosis maintained bone mass and strength First Online: 18 September 2015 DOI: 10.1007/s00198-015-3308-6 Osteoporos Int (2016) 27: 283. doi:10.1007/s00198-015-3308-6

Dai WW, EYA Lay, L Jiang, M Goldspring, S Amugongo, GQ Jin, N Dave, RO Ritchie, Yao W *. Beta-ecdysone prevents glucocorticoid-inhibitions on bone formation and bone strength. Bone 74: 48-57; 2015

Dai WW, EYA Lay, Gq Jin, QJ Xing, L Jiang, HL Chen, RO. Ritchie, NE. Lane, Yao W* Sexual Difference In Peak Bone Mass Regulation: Potential Treatment Option. Clinical Orthopedic Related Research 473:2495-2504; 2015

Yao W *, WW Dai, EYA Lay, L Jiang, N Dave, RO Ritchie, XD Li, HZ Ke, NE Lane Effects of Sclerostin antibody on osteoblast and osteocyte viability/autophagy in mouse model of glucocorticoid-induced bone loss. Osteoporosis International 27:283-294, 2016

Lay EYA, Kot A, Liu RW, Zhang HL, Chen HY, Lam K, Lane NE, Yao W*. Improved mobilization of exogenous mesenchymal stem cells to bone for fracture healing: paracrine effects and sex differences. Stem Cells 34:2587-2600, 2016.

Mohan G, Lay EYA, Berka H, Ringwood L, Kot A, Chen HY, Yao, W, Lane NE. A novel hybrid compound LLP2A-Ale both prevented and rescued the osteoporotic phenotype in a mouse model of glucocorticoid-induced osteoporosis. Calcif Tissue Int. 2016 (on-line)