Mesenchymal Progenitor Cells and Their Orthopedic Applications: Forging a Path towards Clinical Trials

Mesenchymal progenitor cells (MPCs) are nonhematopoietic multipotent cells capable of differentiating into mesenchymal and nonmesenchymal lineages. While they can be isolated from various tissues, MPCs isolated from the bone marrow are best characterized. These cells represent a subset of bone marrow stromal cells (BMSCs) which, in addition to their differentiation potential, are critical in supporting proliferation and differentiation of hematopoietic cells. They are of clinical interest because they can be easily isolated from bone marrow aspirates and expanded in vitro with minimal donor site morbidity. The BMSCs are also capable of altering disease pathophysiology by secreting modulating factors in a paracrine manner. Thus, engineering such cells to maximize therapeutic potential has been the focus of cell/gene therapy to date. Here, we discuss the path towards the development of clinical trials utilizing BMSCs for orthopaedic applications. Specifically, we will review the use of BMSCs in repairing critical-sized defects, fracture nonunions, cartilage and tendon injuries, as well as in metabolic bone diseases and osteonecrosis. A review of www.ClinicalTrials.gov of the United States National Institute of Health was performed, and ongoing clinical trials will be discussed in addition to the sentinel preclinical studies that paved the way for human investigations

Therapeutic Implications of PPAR γ

Osteosarcoma (OS) is the most common nonhematologic malignancy of bone in children and adults. Although dysregulation of tumor suppressor genes and oncogenes, such as Rb, p53, and the genes critical to cell cycle control, genetic stability, and apoptosis have been identified in OS, consensus genetic changes that lead to OS development are poorly understood. Disruption of the osteogenic differentiation pathway may be at least in part responsible for OS tumorigenesis. Current OS management involves chemotherapy and surgery. Peroxisome proliferator-activated receptor (PPAR) agonists and/or retinoids can inhibit OS proliferation and induce apoptosis and may inhibit OS growth by promoting osteoblastic terminal differentiation. Thus, safe and effective PPAR agonists and/or retinoid derivatives can be then used as adjuvant therapeutic drugs for OS therapy. Furthermore, these agents have the potential to be used as chemopreventive agents for the OS patients who undergo the resection of the primary bone tumors in order to prevent local recurrence and/or distal pulmonary metastasis

Lysophosphatidic Acid Acyltransferase β (LPAATβ) Promotes the Tumor Growth of Human Osteosarcoma

Osteosarcoma is the most common primary malignancy of bone with poorly characterized molecular pathways important in its pathogenesis. Increasing evidence indicates that elevated lipid biosynthesis is a characteristic feature of cancer. We sought to investigate the role of lysophosphatidic acid acyltransferase β (LPAATβ, aka, AGPAT2) in regulating the proliferation and growth of human osteosarcoma cells. LPAATβ can generate phosphatidic acid, which plays a key role in lipid biosynthesis as well as in cell proliferation and survival. Although elevated expression of LPAATβ has been reported in several types of human tumors, the role of LPAATβ in osteosarcoma progression has yet to be elucidated.Endogenous expression of LPAATβ in osteosarcoma cell lines is analyzed by using semi-quantitative PCR and immunohistochemical staining. Adenovirus-mediated overexpression of LPAATβ and silencing LPAATβ expression is employed to determine the effect of LPAATβ on osteosarcoma cell proliferation and migration in vitro and osteosarcoma tumor growth in vivo. We have found that expression of LPAATβ is readily detected in 8 of the 10 analyzed human osteosarcoma lines. Exogenous expression of LPAATβ promotes osteosarcoma cell proliferation and migration, while silencing LPAATβ expression inhibits these cellular characteristics. We further demonstrate that exogenous expression of LPAATβ effectively promotes tumor growth, while knockdown of LPAATβ expression inhibits tumor growth in an orthotopic xenograft model of human osteosarcoma.Our results strongly suggest that LPAATβ expression may be associated with the aggressive phenotypes of human osteosarcoma and that LPAATβ may play an important role in regulating osteosarcoma cell proliferation and tumor growth. Thus, targeting LPAATβ may be exploited as a novel therapeutic strategy for the clinical management of osteosarcoma. This is especially attractive given the availability of selective pharmacological inhibitors

Retinoic Acids Potentiate BMP9-Induced Osteogenic Differentiation of Mesenchymal Progenitor Cells

As one of the least studied bone morphogenetic proteins (BMPs), BMP9 is one of the most osteogenic BMPs. Retinoic acid (RA) signaling is known to play an important role in development, differentiation and bone metabolism. In this study, we investigate the effect of RA signaling on BMP9-induced osteogenic differentiation of mesenchymal progenitor cells (MPCs).Both primary MPCs and MPC line are used for BMP9 and RA stimulation. Recombinant adenoviruses are used to deliver BMP9, RARalpha and RXRalpha into MPCs. The in vitro osteogenic differentiation is monitored by determining the early and late osteogenic markers and matrix mineralization. Mouse perinatal limb explants and in vivo MPC implantation experiments are carried out to assess bone formation. We find that both 9CRA and ATRA effectively induce early osteogenic marker, such as alkaline phosphatase (ALP), and late osteogenic markers, such as osteopontin (OPN) and osteocalcin (OC). BMP9-induced osteogenic differentiation and mineralization is synergistically enhanced by 9CRA and ATRA in vitro. 9CRA and ATRA are shown to induce BMP9 expression and activate BMPR Smad-mediated transcription activity. Using mouse perinatal limb explants, we find that BMP9 and RAs act together to promote the expansion of hypertrophic chondrocyte zone at growth plate. Progenitor cell implantation studies reveal that co-expression of BMP9 and RXRalpha or RARalpha significantly increases trabecular bone and osteoid matrix formation.Our results strongly suggest that retinoid signaling may synergize with BMP9 activity in promoting osteogenic differentiation of MPCs. This knowledge should expand our understanding about how BMP9 cross-talks with other signaling pathways. Furthermore, a combination of BMP9 and retinoic acid (or its agonists) may be explored as effective bone regeneration therapeutics to treat large segmental bony defects, non-union fracture, and/or osteoporotic fracture

Proximal Junctional Kyphosis: Inter- and Intraobserver Reliability of Radiographic Measurements in Adult Spinal Deformity.

STUDY DESIGN: Reliability study of radiographic measures of proximal junctional kyphosis (PJK) in patients with adult spinal deformity (ASD). OBJECTIVE: To assess impacts of level of proximal endpoint and vertebral fracture on reliability of measurement of junctional kyphosis. SUMMARY OF BACKGROUND DATA: Radiographic assessment is important in determining management of patients with PJK or proximal junctional failure (PJF). No study to date has evaluated the reliability of radiographic measurement of the junctional kyphotic angle after surgery for ASD. METHODS: Postoperative radiographs from 52 patients with ASD were divided into four categories based on the level of the upper instrumented vertebra (UIV) and the presence or absence of PJF: upper thoracic without failure (UT), thoracolumbar without failure (TL), upper thoracic with PJF (UTF), and thoracolumbar with PJF (TLF). Nine surgeon reviewers performed radiographic measurements of kyphosis between UIV+2 and UIV twice at least 4 weeks apart. Intraclass correlation coefficients (ICC) were calculated to determine inter- and intraobserver reliability. RESULTS: Interobserver reliability for measurements of UT, TL, UTF, and TLF were all almost perfect with ICC scores of 0.917, 0.965, 0.956, and 0.882, and 0.932, 0.975, 0958, and 0.989, for sessions 1 and 2, respectively. Similarly, ICCs for kyphosis measurements for the TL and TLF group had almost perfect agreement with means of 0.898 (range: 0.817-0.969) and 0.976 (range: 0.931-0.995), respectively. ICCs for measurements for the UT and UTF groups all had substantial or almost perfect agreement with means of 0.801 (range: 0.662-0.942) and 0.879 (range: 0.760-0.988), respectively. CONCLUSION: The present study demonstrates high inter- and intraobserver reliability of PJK measurement following instrumented fusion for ASD, independent of the presence or absence of PJF. Although slightly lower for upper thoracic than for thoracolumbar proximal endpoints, all ICCs consistently reached at least substantial agreement and near perfect agreement for most. LEVEL OF EVIDENCE: 4

Mesenchymal stem cells: Molecular characteristics and clinical applications

Mesenchymal stem cells (MSCs) are non-hematopoietic stem cells with the capacity to differentiate into tissues of both mesenchymal and non-mesenchymal origin. MSCs can differentiate into osteoblastic, chondrogenic, and adipogenic lineages, although recent studies have demonstrated that MSCs are also able to differentiate into other lineages, including neuronal and cardiomyogenic lineages. Since their original isolation from the bone marrow, MSCs have been successfully harvested from many other tissues. Their ease of isolation and ex vivo expansion combined with their immunoprivileged nature has made these cells popular candidates for stem cell therapies. These cells have the potential to alter disease pathophysiology through many modalities including cytokine secretion, capacity to differentiate along various lineages, immune modulation and direct cell-cell interaction with diseased tissue. Here we first review basic features of MSC biology including MSC characteristics in culture, homing mechanisms, differentiation capabilities and immune modulation. We then highlight some in vivo and clinical evidence supporting the therapeutic roles of MSCs and their uses in orthopedic, autoimmune, and ischemic disorders

Construction and characterization of recombinant adenovirus that expresses mouse LPAATβ or knocks down human LPAATβ.

<p><b>A</b>. Schematic representation of adenoviral shuttle vector that overexpresses LPAATβ driven by CMV promoter (for making AdR-LPAATβ). The shuttle vector also expresses RFP marker. <b>B</b>. Schematic representation of adenoviral pSOS shuttle vector that expresses siRNA driven by dual opposing promoters (for making AdR-siLPAATβ). The pSOS shuttle vector also expresses RFP marker. The four siRNA targeting sites against human LPAATβ are listed. <b>C</b>. Adenovirus-mediated effective transduction of human osteosarcoma cells. Optimal titer of the recombinant adenoviruses AdR-LPAATβ and AdR-siLPAATβ were used to infect 143B cells. The expression of red fluorescent protein (RFP) was examined at 48 h after infection. <b>D</b>. Characterization of AdR-LPAATβ-mediated overxpression and AdR-siLPAATβ-mediated knockdown. Subconfluent 143B cells were infected with AdR-LPAATβ, AdR-siLPAATβ, or AdRFP control for 48 h. Total RNA were collected and subjected to RT-PCR. The resultant cDNA was subjected to semi-quantitative PCR using primers specific for mouse LPAATβ mRNA (for AdR-LPAATβ infection) or human LPAATβ mRNA (for AdR-LPAATβ infection). GAPDH was used as an internal control to normalize all samples.</p