Transplanted bone marrow mononuclear cells and MSCs impart clinical benefit to children with osteogenesis imperfecta through different mechanisms.

Transplantation of whole bone marrow (BMT) as well as ex vivo-expanded mesenchymal stromal cells (MSCs) leads to striking clinical benefits in children with osteogenesis imperfecta (OI); however, the underlying mechanism of these cell therapies has not been elucidated. Here, we show that non-(plastic)-adherent bone marrow cells (NABMCs) are more potent osteoprogenitors than MSCs in mice. Translating these findings to the clinic, a T cell-depleted marrow mononuclear cell boost (> 99.99% NABMC) given to children with OI who had previously undergone BMT resulted in marked growth acceleration in a subset of patients, unambiguously indicating the therapeutic potential of bone marrow cells for these patients. Then, in a murine model of OI, we demonstrated that as the donor NABMCs differentiate to osteoblasts, they contribute normal collagen to the bone matrix. In contrast, MSCs do not substantially engraft in bone, but secrete a soluble mediator that indirectly stimulates growth, data which provide the underlying mechanism of our prior clinical trial of MSC therapy for children with OI. Collectively, our data indicate that both NABMCs and MSCs constitute effective cell therapy for OI, but exert their clinical impact by different, complementary mechanisms. The study is registered at www.clinicaltrials.gov as NCT00187018.

Transient activation of Wnt/{beta}-catenin signaling induces abnormal growth plate closure and articular cartilage thickening in postnatal mice.

Wnt/beta-catenin signaling is required for skeletal development and organization and for function of the growth plate and articular cartilage. To further clarify these roles and their possible pathophysiological importance, we created a new transgenic mouse model in which Wnt/beta-catenin signaling can be activated in cartilage for specific periods of time. These transgenic mice expressed a constitutive active form of beta-catenin fused to a modified estrogen receptor ligand-binding domain under the control of cartilage-specific collagen 11alpha2 promoter/enhancer. Transient Wnt/beta-catenin signaling activation in young adult mice by tamoxifen injections induced growth retardation and severe deformities in knee joints. Tibial and femoral growth plates displayed an excessive number of apoptotic cells and eventually underwent abnormal regression. Articular cartilage exhibited an initial acute loss of proteoglycan matrix that was followed by increases in thickness, cell density, and cell proliferation. In reciprocal studies, we found that conditional ablation of beta-catenin in postnatal mice using a Col2-CreER strategy led to hypocellularity in articular cartilage, growth plate disorganization, and a severe reduction in bone volume. Together, these data provide evidence that Wnt/beta-catenin signaling has important and distinct roles in growth plate and articular cartilage and that postnatal dysregulation of this signaling pathway causes diverse structural and functional changes in the two cartilaginous structures.

Oxytocin deficiency impairs maternal skeletal remodeling.

We have reported that the posterior pituitary hormone, oxytocin (OT), known for its effects in inducing parturition, lactation and social bonding, is also a skeletal hormone. Here, we demonstrate that OT plays a key role in enabling maternal skeletal mobilization during pregnancy by enhancing the formation of bone resorbing osteoclasts. Osteoclast formation ex vivo is thus diminished in pregnant mothers with genetic OT-deficiency. OT(-/-) pups at day E20 also show a defect in trabecular bone. microCT measurements reveal normal bone volume, but increased trabecular numbers, suggesting that trabeculae in OT(-/-) pups are hypomineralized. We suggest that OT facilitates intergenerational transfer of calcium ions from a pregnant mother to the pups.

Promotion of hydroxyapatite-associated, stem cell-based bone regeneration by CCN2.

Multiple roles have been already recognized for CCN2 in cartilage development and regeneration. However, the effects of CCN2 on bone regeneration remain to be elucidated. In this study, the utility of CCN2 on bone regeneration was examined in vitro and in vivo in combination with hydroxyapatite (HAp) as a scaffold. Human bone marrow stromal cells (hBMSCs) were isolated from human iliac bone marrow aspirates of healthy donors and expanded, and the effects of CCN2 on their proliferation and migration were examined in vitro. The proliferation of hBMSCs on a plastic or HAp plate was significantly enhanced by CCN2. Moreover, the migration of hBMSCs also dramatically increased by CCN2. Interestingly, a C-terminal signal modular fragment of CCN2 (CT-module) also enhanced the cell proliferation and migration as efficiently as the full-length CCN2. Next, in order to estimate the effect of CCN2 on the migration and survival of hBMSCs and bone formation inside the HAp scaffold in vivo, two experiments were performed. First, the porous HAp carrier was cultured with hBMSCs for a week, and the cell-scaffold hybrid was transplanted with or without CCN2 subcutaneously into immunocompromised mice. CCN2 accelerated the hBMSC-like cell migration and survival inside the porous HAp within 4 weeks after transplantation. Second, the porous HAp carrier with or without CCN2 was directly implanted into bone defects within a rabbit mandible, and bone regeneration inside was evaluated. As a result, CCN2 efficiently induced the cell invasion and bone formation inside the porous HAp scaffold. These findings suggest that CCN2 and its CT-module fragment could be useful for regeneration and reconstruction of large-scale bone defects.

Intervertebral disc development is regulated by Wnt/ß-catenin signaling.

STUDY DESIGN: Histologic analysis of intervertebral disc (IVD) in three types of transgenic mice. OBJECTIVE: To investigate the role of Wnt/ß-catenin signaling in regulation of IVD development and organization. SUMMARY OF BACKGROUND DATA: ß-catenin dependent Wnt signaling is one of the central regulators in cartilage development during limb skeletal formation. Little is known, however, about the physiologic relevance of this signaling pathway to IVD development and organization. METHODS: Temporal-spatial distribution of Wnt/ß-catenin signaling activity was examined in IVD using Wnt/ß-catenin reporter (TOPGAL) mice. The structural changes in the mouse IVD components such as the nucleus pulposus (NP), endplate (EP), annulus fibrosus (AF), and the growth plate (GP) of the vertebral body were analyzed after transient activation of Wnt/ß-catenin signaling or deletion of ß-catenin in the mice. RESULTS: Activity of Wnt/ß-catenin signaling was high in EP, AF, and GP in the embryonic stages and decreased at the postnatal stage; it was undetectable in the embryonic NP but upregulated after birth. The transient activation of Wnt/ß-catenin signaling caused severe deterioration of the GP and the AF, whereas deficiency of ß-catenin accelerated bone formation in between EP and GP. CONCLUSION: The findings in this study suggest that proper regulation of Wnt/ß-catenin signaling is required for development and organization of IVD.

Potent inhibition of heterotopic ossification by nuclear retinoic acid receptor-γ agonists.

Heterotopic ossification consists of ectopic bone formation within soft tissues after surgery or trauma. It can have debilitating consequences, but there is no definitive cure. Here we show that heterotopic ossification was essentially prevented in mice receiving a nuclear retinoic acid receptor-γ (RAR-γ) agonist. Side effects were minimal, and there was no significant rebound effect. To uncover the mechanisms of these responses, we treated mouse mesenchymal stem cells with an RAR-γ agonist and transplanted them into nude mice. Whereas control cells formed ectopic bone masses, cells that had been pretreated with the RAR-γ agonist did not, suggesting that they had lost their skeletogenic potential. The cells became unresponsive to rBMP-2 treatment in vitro and showed decreases in phosphorylation of Smad1, Smad5 and Smad8 and in overall levels of Smad proteins. In addition, an RAR-γ agonist blocked heterotopic ossification in transgenic mice expressing activin receptor-like kinase-2 (ALK2) Q207D, a constitutively active form of the receptor that is related to ALK2 R206H found in individuals with fibrodysplasia ossificans progressiva. The data indicate that RAR-γ agonists are potent inhibitors of heterotopic ossification in mouse models and, thus, may also be effective against injury-induced and congenital heterotopic ossification in humans.

Inhibition of ectopic bone formation by a selective retinoic acid receptor alpha-agonist: a new therapy for heterotopic ossification?

Heterotopic ossification (HO) consists of formation of ectopic cartilage followed by endochondral bone and is triggered by major surgeries, large wounds, and other conditions. Current therapies, including low-dose irradiation, are not always effective and do not target the skeletogenic process directly. Because chondrogenesis requires a decrease of nuclear retinoic acid receptor alpha (RARalpha) action, we reasoned that pharmacologic activation of this receptor pathway should inhibit HO. Thus, we selected the synthetic retinoid NRX195183, a potent and highly selective RARalpha-agonist, and found that it did inhibit chondrogenesis in mouse limb micromass cultures. We established a mouse HO model consisting of subcutaneous implantation of Matrigel mixed with rhBMP-2. Control mice receiving daily oral doses of vehicle (peanut oil) or retinol (a natural nonactive retinoid precursor) developed large HO-like masses by days 9-12 that displayed abundant cartilage, endochondral bone, vessels, and marrow. In contrast, formation of HO-like masses was markedly reduced in companion mice receiving daily oral doses of alpha-agonist. These ectopic masses contained sharply reduced amounts of cartilage and bone, blood vessels, and TRAP-positive osteoclasts, and expressed markedly lower levels of master chondrogenic genes including Sox9, cartilage genes such as collagen XI and X, and osteogenic genes including Runx2. The data provide proof-of-principle evidence that a pharmacological strategy involving a selective RARalpha-agonist can indeed counteract an ectopic skeletal-formation process effectively and efficiently, and could thus represent a novel preventive treatment for HO.