Daniel Lozano

Bone fracture healing: Cell therapy in delayed unions and nonunions

Bone fracture healing impairment related to mechanical problems has been largely corrected by advances in fracture management. Better protocols, more strict controls of time and function, and hardware and surgical technique evolution have contributed to better prognosis, even in complex fractures. However, atrophic nonunion persists in clinical cases where, for different reasons, the osteogenic capability is impaired. When this is the case, a better understanding of the basic mechanisms under bone repair and augmentation techniques may put in perspective the current possibilities and future opportunities. Among those, cell therapy particularly aims to correct this insufficient osteogenesis. However, the launching of safe and efficacious cell therapies still requires substantial amount of research, especially clinical trials. This review will envisage the current clinical trials on bone healing augmentation based on cell therapy, with the experience provided by the REBORNE Project, and the insight from investigator-driven clinical trials on advanced therapies towards the future. This article is part of a Special Issue entitled Stem Cells and Bone.

The osteoinductive properties of mesoporous silicate coated with osteostatin in a rabbit femur cavity defect model.

Parathyroid hormone-related protein (PTHrP) is an important regulator of bone formation and remodeling. Our recent findings demonstrate that PTHrP (107-111) (osteostatin) loaded onto silica-based ordered mesoporous SBA15 materials exhibit osteogenic features in osteoblastic cell cultures. We aimed here to elucidate whether these peptide-coated materials might be suitable for promoting bone repair following a cavitary defect in the rabbit femur. Histological examination revealed the absence of significant inflammation or bone resorption within the time of study (4 and 8 weeks) after implantation. At 8 weeks, the peptide-unloaded materials were still separated from the bone marrow by a fibrous cap, which was greatly diminished by the presence of the PTHrP peptide. By using μCT analysis, new bone formation was evident at different distances from the implants, mainly for the latter peptide-loaded biomaterials. This was confirmed by performing immunostaining for different osteoblast markers. Our findings demonstrate that these PTHrP (107-111)-loaded bioceramics significantly improve local bone induction, as compared to that observed with the unloaded material.

Role of parathyroid hormone-related protein in the decreased osteoblast function in diabetes-related osteopenia.

A deficit in bone formation is a major factor in diabetes-related osteopenia. We examined here whether diabetes-associated changes in osteoblast phenotype might in part result from a decrease in PTH-related protein (PTHrP). We used a bone marrow ablation model in diabetic mice by multiple streptozotocin injections. PTHrP (1-36) (100 microg/kg, every other day) or vehicle was administered to mice for 13 d starting 1 wk before marrow ablation. Diabetic mice showed bone loss in both the intact femur and the regenerating tibia on d 6 after ablation; in the latter, this was related to decreased bone-forming cells, osteoid surface, and blood vessels, and increased marrow adiposity. Moreover, a decrease in matrix mineralization occurred in ex vivo bone marrow cultures from the unablated tibia from diabetic mice. These skeletal alterations were associated with decreased gene expression (by real-time PCR) of Runx2, osterix, osteocalcin, PTHrP, the PTH type 1 receptor, vascular endothelial growth factor and its receptors, and osteoprotegerin to receptor activator of nuclear factor-kappaB ligand mRNA ratio, and increased peroxisome proliferator-activated receptor-gamma2 mRNA levels. Similar changes were induced by hyperosmotic (high glucose or mannitol) medium in osteoblastic MC3T3-E1 cells, which were mimicked by adding a neutralizing anti-PTHrP antibody or PTH type 1 receptor antagonists to these cells in normal glucose medium. PTHrP (1-36) administration reversed these changes in both intact and regenerating bones from diabetic mice in vivo, and in MC3T3-E1 cells exposed to high glucose. These findings strongly suggest that PTHrP has an important role in the altered osteoblastic function related to diabetes.

GLP-1 and exendin-4 can reverse hyperlipidic-related osteopenia

Increased fat mass contributes to bone deterioration. Glucagon-like peptide 1 (GLP-1) and its related peptide exendin 1-39 amide (Ex-4), two lipid-lowering peptides, exert osteogenic effects in diabetic states. We examined the actions of 3-day administration of GLP-1 or Ex-4 on bone remodeling markers and on bone mass and structure in hyperlipidic (HL) and hypercaloric rats. Wistar rats on a hyperlipidemic diet for 35 days were subcutaneously administered GLP-1 (0.86  nmol/kg per h), Ex-4 (0.1  nmol/kg per h), or saline (control) by continuous infusion for 3 days. After killing, tibiae were removed for total RNA and protein isolation, as well as femurs and L1-L4 vertebrae for bone mass and quality assessment. Body weight and plasma insulin were unaltered in HL rats, which showed osteopenia (by dual-energy X-ray absorptiometry), associated with hyperglycemia, hypertriglyceridemia, and hypercholesterolemia. GLP-1 or Ex-4 administration decreased the levels of glucose, triglycerides, and total cholesterol in plasma but increased osteocalcin (OC) gene expression and the osteoprotegerin (OPG)/receptor activator of NF-κB ligand (RANKL) ratio - at the expense of an augmented OPG - above corresponding control values in the tibia. Each tested peptide similarly reversed the decreased femoral and vertebral bone mass in these rats, whereas the deteriorated trabecular structure in the vertebrae improved associated with normalization of bone remodeling. These findings demonstrate that GLP-1 and Ex-4 are similarly efficient in reversing the bone alterations in this HL rat model, which has proven to be useful for studying the fat-bone relationships.

Comparison of the skeletal effects induced by daily administration of PTHrP (1-36) and PTHrP (107-139) to ovariectomized mice.

We here compared the changes induced by subcutaneous injection of PTHrP (1–36) or PTHrP (107–139) (80 µg/kg/day, 5 days/week for 4 or 8 weeks) in bone histology and bone remodeling factors, and in bone marrow cells (BMCs) ex vivo, in ovariectomized (OVX) mice. We also examined the osteogenic effects of these peptides in mouse mesenchymal C3H10T1/2 cells under oxidative stress condition in vitro, which recapitulates the effects of OVX. We confirmed that PTHrP (1–36) exerts bone anabolic actions, as assessed by bone histology and osteoblast differentiation markers in the long bones and plasma from OVX mice. PTHrP (107–139) was also efficient in stimulating several bone formation parameters, and it dramatically decreased bone resorption markers. Moreover, both PTHrP peptides modulate DKK‐1 and Sost/sclerostin in osteoblast‐like UMR‐106 cells highly expressing these Wnt pathway inhibitors, related to their osteogenic action in this in vivo scenario. Administration of either PTHrP peptide improved osteogenic differentiation in BMCs from OVX mice ex vivo and in mouse mesenchymal C3H10T1/2 cells under oxidative stress condition in vitro. These data demonstrate that PTHrP (1–36) and PTHrP (107–139) can exert similar osteogenic effects in the appendicular skeleton of OVX mice. Our results suggest that these effects might occur in part by modulating the Wnt pathway. These findings lend credence to the notion that the osteogenic action of PTHrP (107–139) is likely a consequence of its anti‐resorptive and anabolic features, and further support the usefulness of PTHrP (1–36) as a bone anabolic peptide in the setting of estrogen‐depletion. J. Cell. Physiol. 227: 1752–1760, 2012.

Comparison of the osteoblastic activity conferred on Si-doped hydroxyapatite scaffolds by different osteostatin coatings

Parathyroid hormone-related protein (107-111) (osteostatin) induces osteogenic effects in osteoblasts in vitro and in regenerating bone in mice and rabbits. In this study we used osteoblastic MC3T3-E1 cell cultures to evaluate and compare the bioactivity of this peptide either adsorbed or covalently bound (by its C-terminus) to Si-doped hydroxyapatite (Si-HA) scaffolds after organic (-NH(2)) functionalization. By these means osteostatin can be locally released or kept anchored to the scaffold surface. This was confirmed by chemical analysis and by testing the efficiency of osteostatin-loaded Si-HA scaffolds (placed in Transwell chambers) in healing a scratch wound in mouse pluripotent mesenchymal C3H10T1/2 cells. Our results show that exposure of MC3T3-E1 cell monolayers to Si-HA scaffolds with both types of osteostatin coating (deliverable or immobilized), in contrast to those without peptide, similarly stimulated cell growth and matrix mineralization. These findings demonstrate that osteostatin release from Si-HA scaffolds is not essential to promote osteoblastic growth and function in vitro, and lend credence to considering osteostatin a bone regenerating factor.

Osteostatin improves the osteogenic activity of fibroblast growth factor-2 immobilized in Si-doped hydroxyapatite in osteoblastic cells.

Si-doped hydroxyapatite (Si-HA) is a suitable ceramic for the controlled release of agents to improve bone repair. We recently showed that parathyroid hormone-related protein (PTHrP) (107-111) (osteostatin) has remarkable osteogenic features in various in vitro and in vivo systems. Fibroblast growth factor (FGF)-2 modulates osteoblastic function and induces angiogenesis, and can promote osteoblast adhesion and proliferation after immobilization on Si-HA. In the present study we examined whether osteostatin might improve the biological efficacy of FGF-2-coated Si-HA in osteoblastic MC3T3-E1 cells in vitro. We found that Si-HA/FGF-2 in the presence or absence of osteostatin (100 nM) similarly increased cell growth (by about 50%). However, addition of the latter peptide to Si-HA/FGF-2 significantly enhanced gene expression of Runx2, osteocalcin, vascular endothelial growth factor (VEGF) and the VEGF receptors 1 and 2, without significantly affecting that of FGF receptors in these cells. Moreover, secreted VEGF in the MC3T3-E1 cell conditioned medium, which induced the proliferation of pig endothelial-like cells, was also enhanced by these combined factors. The synergistic action of osteostatin and Si-HA/FGF-2 on the VEGF system was abrogated by a mitogen-activated protein kinase inhibitor (U0126) and by the calcium antagonist verapamil. This action was related to an enhancement of alkaline phosphatase activity and matrix mineralization in MC3T3-E1 cells, and also in primary human osteoblastic cells. These in vitro data show that osteostatin increases the osteogenic efficacy of a Si-HA/FGF-2 biomaterial by a mechanism involving mitogen-activated protein kinases and intracellular Ca(2+). These findings provide an attractive strategy for bone tissue engineering.

Osteostatin-loaded onto mesoporous ceramics improves the early phase of bone regeneration in a rabbit osteopenia model.

Parathyroid hormone-related protein (PTHrP) is an important modulator of bone formation. Recently, we reported that PTHrP (107-111) (osteostatin) coating onto mesoporous ceramics confers osteogenic activity to these materials. Bone repair is dramatically compromised in osteopenia/osteoporosis. Thus, we examined the efficacy of unmodified and organically modified SBA15 ceramics loaded with osteostatin in promoting bone repair in an osteoporotic rabbit model. Osteoporosis was induced in New Zealand rabbits by methylprednisolone administration, and healthy rabbits were used as controls. Tested materials were implanted into a femoral cavitary defect, and animals were sacrificed at 2 weeks post-implantation. At this time, implants were encapsulated by a variable layer of fibrotic tissue with no evidence of inflammation. Similarly to observations in normal rabbits, both types of osteostatin-loaded bioceramics induced tissue regeneration associated with increased staining for PCNA, Runx2, osteopontin, and/or vascular endothelial growth factor in osteoporotic rabbits. Our present findings demonstrate that these osteostatin-bearing bioceramics increase the early repair response not only in normal bone but also in osteoporotic bone after a local injury.

Inhibition of the canonical Wnt pathway by high glucose can be reversed by parathyroid hormone‐related protein in osteoblastic cells

Recent in vivo findings suggest that the bone sparing effect of parathyroid hormone‐related protein (PTHrP) in diabetic mice might occur at least in part through targeting a suppressed Wnt/β‐catenin pathway in osteoblasts. We here aimed to examine the inhibitory action of a high glucose environment on specific components of the canonical Wnt pathway, and the putative compensatory effects of PTHrP, in osteoblastic cell cultures. Mouse osteoblastic MC3T3‐E1 cells and primary cultures of fetal mouse calvaria were exposed to normal (5.5 mM) or high (25 mM) D‐glucose (HG), with or without PTHrP (1–36) or PTHrP (107–139) for different times. In some experiments, MC3T3‐E1 cells were incubated with the Wnt pathway activators Wnt3a and LiCl, or were transfected with plasmids encoding either a mutated β‐catenin that cannot be targeted for degradation or a human PTHrP (−36/+139) cDNA, or the corresponding empty plasmid, in the presence or absence of HG. The gene expression of Wnt3a and low density receptor‐like proteins (LRP)‐5 and 6, as well as β‐catenin protein stabilization and β‐catenin‐dependent transcription activity were evaluated. Oxidative stress status under HG condition was also assessed. The present data demonstrate that HG can target different components of the canonical Wnt pathway, while β‐catenin degradation appears to be a key event leading to inhibition of Wnt/β‐catenin signaling in mouse osteoblastic cells. Both PTHrP peptides tested were able to counteract this deleterious action of HG. These in vitro findings also provide new clues to understand the underlying mechanisms whereby PTHrP can increase bone formation. J. Cell. Biochem. 114: 1908–1916, 2013.

Functional Roles of the Nuclear Localization Signal of Parathyroid Hormone-Related Protein (PTHrP) in Osteoblastic Cells

PTHrP is an important regulator of bone remodelling, apparently by acting through several sequence domains. We here aimed to further delineate the functional roles of the nuclear localization signal (NLS) comprising the 88-107 amino acid sequence of PTHrP in osteoblasts. PTHrP mutants from a human PTHrP (-36/+139) cDNA (wild type) cloned into pcDNA3.1 plasmid with deletion (Δ) of the signal peptide (SP), NLS, T(107), or T107A replacing T(107) by A(107) were generated and stably transfected into osteoblastic MC3T3-E1 cells. In these cells, intracellular trafficking, cell proliferation and viability, as well as cell differentiation were evaluated. In these transfected cells, PTHrP was detected in the cytoplasm and also in the nucleus, except in the NLS mutant. Meanwhile, the PTH type 1 receptor (PTH1R) accumulates in the cytoplasm except for the ΔSP mutant in which the receptor remains at the cell membrane. PTHrP-wild type cells showed enhanced growth and viability, as well as an increased matrix mineralization, alkaline phosphatase activity, and osteocalcin gene expression; and these features were inhibited or abolished in ΔNLS or ΔT(107) mutants. Of note, these effects of PTHrP overexpression on cell growth and function were similarly decreased in the ΔSP mutant after PTH1R small interfering RNA transfection or by a PTH1R antagonist. The present in vitro findings suggest a mixed model for PTHrP actions on osteoblastic growth and function whereby this protein needs to be secreted and internalized via the PTH1R (autocrine/paracrine pathway) before NLS-dependent shuttling to the nucleus (intracrine pathway).