Graphene-Oxide Porous Biopolymer Hybrids Enhance In Vitro Osteogenic Differentiation and Promote Ectopic Osteogenesis In Vivo.

Over the years, natural-based scaffolds have presented impressive results for bone tissue engineering (BTE) application. Further, outstanding interactions have been observed during the interaction of graphene oxide (GO)-reinforced biomaterials with both specific cell cultures and injured bone during in vivo experimental conditions. This research hereby addresses the potential of fish gelatin/chitosan (GCs) hybrids reinforced with GO to support in vitro osteogenic differentiation and, further, to investigate its behavior when implanted ectopically. Standard GCs formulation was referenced against genipin (Gp) crosslinked blend and 0.5 wt.% additivated GO composite (GCsGp/GO 0.5 wt.%). Pre-osteoblasts were put in contact with these composites and induced to differentiate in vitro towards mature osteoblasts for 28 days. Specific bone makers were investigated by qPCR and immunolabeling. Next, CD1 mice models were used to assess de novo osteogenic potential by ectopic implantation in the subcutaneous dorsum pocket of the animals. After 4 weeks, alkaline phosphate (ALP) and calcium deposits together with collagen synthesis were investigated by biochemical analysis and histology, respectively. Further, ex vivo materials were studied after surgery regarding biomineralization and morphological changes by means of qualitative and quantitative methods. Furthermore, X-ray diffraction and Fourier-transform infrared spectroscopy underlined the newly fashioned material structuration by virtue of mineralized extracellular matrix. Specific bone markers determination stressed the osteogenic phenotype of the cells populating the material in vitro and successfully differentiated towards mature bone cells. In vivo results of specific histological staining assays highlighted collagen formation and calcium deposits, which were further validated by micro-CT. It was observed that the addition of 0.5 wt.% GO had an overall significant positive effect on both in vitro differentiation and in vivo bone cell recruitment in the subcutaneous region. These data support the GO bioactivity in osteogenesis mechanisms as being self-sufficient to elevate osteoblast differentiation and bone formation in ectopic sites while lacking the most common osteoinductive agents.

Mechanical stress affects methylation pattern of GNAS isoforms and osteogenic differentiation of hAT-MSCs.

Mechanical stress exerts a substantial role on skeletal-cell renewal systems, whereas accumulating evidence suggests that epigenetic mechanisms induce changes and differential gene expression. Although the underlying mechanisms remain to be fully elucidated, our study suggests that the influence of the long term mechanical stimulation elicits epigenetic modifications controlling osteogenic differentiation of human adipose tissue multipotential stromal cells (hAT-MSCs) and contributes to an accelerating in vitro osteogenesis. GNAS imprinting gene acts as a critical regulator of osteoblast differentiation and is implicated in human genetic disorders with pathological formation of ectopic-skeletal bone. Investigating a wide variety of stimuli, we showed that daily mechanical stretch on hAT-MSCs of 7th and 15th days' intervals induced a significant down-regulation in DNA methylation status of critical CpG sites of NESP and GNASXL isoforms, accompanied by up-regulation of the corresponding gene transcripts, and osteogenic differentiation earlier in culture. Importantly, methylation analysis of differentiating bone marrow-derived MSCs revealed similar methylation patterns. Bioinformatic analysis further showed that all CpG islands exhibiting significant methylation alterations encompassed transcriptional repressor CTCF binding sites. We hereby emphasize the need to investigate the epigenetic alterations on hAT-MSCs during environmental mechanical forces and to consider how the knowledge gained through these studies may foster new means of symptoms prevention and management of ectopic bone formation in the clinic.

The Role of Bone Marrow-Derived Cells during Ectopic Bone Formation of Mouse Femoral Muscle in GFP Mouse Bone Marrow Transplantation Model.

Multipotential ability of bone marrow-derived cells has been clarified, and their involvement in repair and maintenance of various tissues has been reported. However, the role of bone marrow-derived cells in osteogenesis remains unknown. In the present study, bone marrow-derived cells during ectopic bone formation of mouse femoral muscle were traced using a GFP bone marrow transplantation model. Bone marrow cells from C57BL/6-Tg (CAG-EGFP) mice were transplanted into C57BL/6 J wild type mice. After transplantation, insoluble bone matrix (IBM) was implanted into mouse muscle. Ectopic bone formation was histologically assessed at postoperative days 7, 14, and 28. Immunohistochemistry for GFP single staining and GFP-osteocalcin double staining was then performed. Bone marrow transplantation successfully replaced hematopoietic cells with GFP-positive donor cells. Immunohistochemical analyses revealed that osteoblasts and osteocytes involved in ectopic bone formation were GFP-negative, whereas osteoclasts and hematopoietic cells involved in bone formation were GFP-positive. These results indicate that bone marrow-derived cells might not differentiate into osteoblasts. Thus, the main role of bone marrow-derived cells in ectopic osteogenesis may not be to induce bone regeneration by differentiation into osteoblasts, but rather to contribute to microenvironment formation for bone formation by differentiating tissue stem cells into osteoblasts.

Alterations of Subchondral Bone Progenitor Cells in Human Knee and Hip Osteoarthritis Lead to a Bone Sclerosis Phenotype.

Subchondral bone tissue plays a key role in the initiation and progression of human and experimental osteoarthritis and has received considerable interest as a treatment target. Elevated bone turnover and remodeling leads to subchondral bone sclerosis that is characterized by an increase in bone material that is less mineralized. The aim of this study was to investigate whether perturbations in subchondral bone-resident progenitor cells might play a role in aberrant bone formation in osteoarthritis. Colony formation assays indicated similar clonogenicity of progenitor cells from non-sclerotic and sclerotic subchondral trabecular bone tissues of osteoarthritic knee and hip joints compared with controls from iliac crest bone. However, the osteogenic potential at the clonal level was approximately two-fold higher in osteoarthritis than controls. An osteogenic differentiation assay indicated an efficient induction of alkaline phosphatase activity but blunted in vitro matrix mineralization irrespective of the presence of sclerosis. Micro-computed tomography and histology demonstrated the formation of de novo calcified tissues by osteoblast-like cells in an ectopic implantation model. The expression of bone sialoprotein, a marker for osteoblast maturation and mineralization, was significantly less in sclerotic progenitor cells. Perturbation of resident progenitor cell function is associated with subchondral bone sclerosis and may be a treatment target for osteoarthritis.

Osteogenic gene array of osteoblasts cultured on a novel osteoinductive biphasic calcium phosphate bone grafting material.

OBJECTIVES: Recently, novel biphasic calcium phosphate (BCP) scaffolds have emerged as a new class of bone grafts with osteoinductive potential demonstrating the ability to form ectopic bone in extra-skeletal sites. The aim of the present study was to perform an osteogenic gene array to target possible genes responsible for eliciting the changes in cell expression responsible for inducing osteoblast differentiation. MATERIALS AND METHODS: Human MG63 osteoblast-like cells were seeded for 24 h on tissue culture plastic or osteoinductive BCP particles and analyzed for upregulated genes using an osteogenesis super-array. Osteoblast-related genes including those transcribed during bone mineralization, bone metabolism, cell growth and differentiation, as well as gene products representing extracellular matrix molecules, transcription factors, and cell adhesion molecules were investigated. RESULTS: An upregulation of genes transcribing biglycan (1.7-fold), bone morphogenetic proteins 1, 2, 4, 6, and 7 (1.5-2.1-fold), various collagen isoforms including 1a1, 1a2, 2a1, and 5a1 (1.73-2.72-fold), colony stimulating factor 2 (2.59-fold), fibroblast growth factor receptor 2 (1.79-fold), fibronectin (2.56-fold), integrin alpha 1, 2, and 3 (1.82-2.24-fold), SOX9 (2.75-fold), transforming growth factor beta receptor 2 (1.72-fold), vitamin D (1.89-fold), and vascular endothelial growth factor A and B (2.00, 1.75-fold) were all significantly (p < 0.05) increased on BCP particles when compared to control tissue culture plastic. CONCLUSION: In summary, a number of activated genes were involved in bone formation following osteoblast attachment to BCP particles. The involvement of key chondrogenic genes hints that bone grafts capable of spontaneously inducing ectopic bone formation may implicate endochondral ossification. Further investigations in the triggered pathways involved in the process of ectopic bone formation are necessary to understand the key inductive properties of these novel osteoinductive BCP particles. CLINICAL RELEVANCE: Novel osteoinductive BCP particles demonstrate a wide range of significant increases over several key molecules implicated in osteogenesis that may be implicated in their ability to form ectopic bone formation.

Bone healing with oxytocin-loaded microporous ß-TCP bone substitute in ectopic bone formation model and critical-sized osseous defect of rat.

AIM: This study investigated the efficacy of the hypothalamic nonapeptide oxytocin (OT) by direct delivery to local defects using a microporous ß-tricalcium phosphate (TCP) as the carrier for the future applications as a method to achieve predictable bone regeneration of large osseous defects requiring sinus bone graft and guided bone regeneration procedures for implant placement. MATERIAL AND METHODS: Both the ectopic and new bone formation induced by the OT-loaded microporous ß-TCP powder was histomorphometrically compared with unloaded ß-TCP in a subcutaneous ectopic bone formation model and calvarial critical-sized defects (CSDs) in 45 rats. RESULTS: The OT-loaded ß-TCP clearly enhanced ectopic bone formation compared with the unloaded control group. A High initial OT dose (250 µg) significantly increased ectopic bone formation at an early healing time-point compared with a lower OT dose (50 µg). The OT-loaded samples displayed greater new bone formation in the rat calvarial CSDs. Extensive new bone formation was achieved in the calvarial CSDs with the higher OT dose. CONCLUSION: These results suggest that local OT delivery to bone substitute promotes new bone formation via an osteoinductive mode of action.

Osseous Metaplasia and Ectopic Bone Formation in a Patient With Hashimoto's Thyroiditis.

Thyroid nodules are frequently accompanied by degenerative changes, such as hemorrhage, cholesterol crystallization, fibrous tissue deposition, or filling with fat. Although calcification is also a common phenomenon, osteogenesis, characterized by mature bone formation, is very rare. Here, we describe a case of Hashimoto's thyroiditis with osseous metaplasia and ectopic bone formation case and discuss its possible causes.

A rare femoral heterotopic bone formation in a 14th-19th century female skeleton from Constância (Portugal).

OBJECTIVE: This paper aims to: (1) document a rare femoral heterotopic ossification (HO), and (2) discuss its aetiology and impact on the individual's locomotion and daily living activities. MATERIALS: Adult female skeleton (SG.14-SK.7) from the village of Constância (Portugal), and dated from the 14th-19th centuries CE. METHODS: The biological profile and the macroscopic analysis of the bone changes were assessed using standardized methods. RESULTS: The macroscopic analysis revealed a large bony mass (8 cm length) located immediately inferior to the small trochanter of the right femur. The lesion exhibited a compact, tubular appearance located at the site of attachment of the pectineus muscle. No signs of bone fracture were observed. CONCLUSIONS: The morphology of the SG.14-SK.7 femoral lesion is compatible with a probable case of myositis ossificans traumatica (MOT), secondary to acute trauma of the pectineus muscle. The underlying trauma episode, such as random accidental and/or occupation-related injury, is unknown. However, it is highly possible that this self-limiting condition significantly impaired the individual's daily life and mobility. SIGNIFICANCE: Evidence of severe acute muscle trauma is a rare finding compared with HO secondary to bone trauma and other minor muscle injuries. Moreover, no cases of MOT affecting the pectineus muscle have been reported in the paleopathological literature to date. LIMITATIONS: Although unlikely, a case of neurogenic or burn-related HO cannot be completely disregarded. It was not possible to undertake radiography as part of this study. SUGGESTIONS FOR FURTHER RESEARCH: The use of imaging techniques to complement the paleopathological description is advised.

Heterotopic Ossification After Arthroscopic Procedures: A Scoping Review of the Literature.

BACKGROUND: Heterotopic ossification (HO) is the formation of bone in soft tissue resultant from inflammatory processes. Lesion formation after arthroscopic procedures is an uncommon but challenging complication. Optimal prophylaxis and management strategies have not been clearly defined. PURPOSE: To present a scoping review of the pathophysiology, risk factors, diagnostic modalities, prophylaxis recommendations, and current treatment practices concerning HO after arthroscopic management of orthopaedic injuries. STUDY DESIGN: Scoping review; Level of evidence, 4. METHODS: A scoping review via a PubMed search was performed according to the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-analyses) guidelines. The search strategy was based on the terms "heterotopic ossification" AND "arthroscopy." The clinical outcomes review included studies on the arthroscopic management of orthopaedic injuries in which the primary subject matter or a secondary outcome was the development of HO. An analysis of the pathophysiology, diagnostic modalities, and management options was reported. RESULTS: A total of 43 studies (33,065 patients) reported on HO after hip arthroscopy, while 21 (83 patients) collectively reported on HO after arthroscopic procedures to the shoulder, elbow, knee, or ankle; however, management techniques were not standardized. Identified risk factors for HO included male sex and mixed impingement pathology, while intraoperative capsular management was not suggested as a contributing factor. Diagnosis of ossification foci was performed using radiography and computed tomography. The rate of HO after hip arthroscopy procedures approached 46% without prophylaxis, and administration of nonsteroidal anti-inflammatory drugs (NSAIDs) decreased occurrence rates to 4% but carries associated risks. External beam radiation has not been exclusively studied for use after arthroscopic procedures. CONCLUSION: HO is a known complication after arthroscopic management of orthopaedic injuries. NSAID prophylaxis has been demonstrated to be effective after hip arthroscopy procedures. Patients with persistent symptoms and mature lesions may be indicated for surgical excision, although variability is present in patient-reported outcome scores postoperatively.

Heterotopic Ossification Following Total Ankle Arthroplasty With Fourth-Generation Prostheses.

BACKGROUND: The purpose of the present study was to assess the radiographic incidence, location, and classification of heterotopic ossification (HO) in patients who underwent total ankle arthroplasty (TAA) with a 4th generation prostheses at a minimum of 1-year follow up. Baseline demographic, radiographic, and operative factors between patents with and without HO were compared. METHODS: Ninety ankles that underwent TAA with a 4th generation protheses, INFINITY (n = 62) or CADENCE (n = 28) were followed for an average of 23.7 (range, 12-49) months. Incidence and location of HO was assessed on weightbearing radiographs, and severity graded according to the modified Brooker classification. Data was compared between patents with and without HO to identity any predisposing factors. RESULTS: In 90 ankles that underwent 4th generation TAA, HO incidence was 55.6% (n = 50); 56.5% (n = 35) for INFINITY, and 53.6% (n = 15) for CADENCE. Twenty-five cases of HO were observed posteriorly, 16 anteriorly, and 9 combined. Severity was as follows; class I in 19 cases (38%), class II in 20 (40%), class III in 9 (18%) and class IV in 2 (4%). A single ankle required a non-revisional reoperation for HO debridement; reoperation rate of 2%. CONCLUSION: The present study suggests a similarly high incidence of HO after TAA with two different 4th generation protheses (INFINITY 56.5%, CADENCE 53.6%). A trend for differences in location and severity between the protheses may also be present. Given the paucity of literature, additional studies with longer follow-up are warranted to discern the significance of HO following TAA with 4th generation protheses. LEVEL OF EVIDENCE: Level III: Retrospective cohort study.

BMSCs and Osteoblast-Engineered ECM Synergetically Promotes Osteogenesis and Angiogenesis in an Ectopic Bone Formation Model.

Bone mesenchymal stem cells (BMSCs) have been extensively used in bone tissue engineering because of their potential to differentiate into multiple cells, secrete paracrine factors, and attenuate immune responses. Biomaterials are essential for the residence and activities of BMSCs after implantation in vivo. Recently, extracellular matrix (ECM) modification with a favorable regenerative microenvironment has been demonstrated to be a promising approach for cellular activities and bone regeneration. The aim of the present study was to evaluate the effects of BMSCs combined with cell-engineered ECM scaffolds on osteogenesis and angiogenesis in vivo. The ECM scaffolds were generated by osteoblasts on the small intestinal submucosa (SIS) under treatment with calcium (Ca)-enriched medium and icariin (Ic) after decellularization. In a mouse ectopic bone formation model, the SIS scaffolds were demonstrated to reduce the immune response, and lower the levels of immune cells compared with those in the sham group. Ca/Ic-ECM modification inhibited the degradation of the SIS scaffolds in vivo. The generated Ca/Ic-SIS scaffolds ectopically promoted osteogenesis according to the results of micro-CT and histological staining. Moreover, BMSCs on Ca/Ic-SIS further increased the bone volume percentage (BV/TV) and bone density. Moreover, angiogenesis was also enhanced by the Ca/Ic-SIS scaffolds, resulting in the highest levels of neovascularization according to the data ofCD31 staining. In conclusion, osteoblast-engineered ECM under directional induction is a promising strategy to modify biomaterials for osteogenesis and angiogenesis. BMSCs synergetically improve the properties of ECM constructs, which may contribute to the repair of large bone defects.

Heterotopic Mesenteric Ossification With Trilineage Hematopoiesis.

Heterotopic ossification (HO) histologically refers to extraskeletal bone formation in non-ossifying tissues, most commonly noted in the extremities, buttocks, abdominal wall, and hip joints. HO developing in the mesentery (heterotopic mesenteric ossification, HMO) is very rare, with fewer than 100 cases reported in the literature. It usually occurs in adult male patients with a history of repeated abdominal trauma. So far, only two cases of HMO have been reported with the development of hematopoietic bone marrow. Here, we report the third case of HMO with true trilineage hematopoiesis in a 66-year-old female with suspicious mesenteric-retained foreign material from prior surgical procedures, including hysterectomy for endometrial adenocarcinoma and multiple repairs for incisional hernia.

Molecular Indicators of Biomaterials Osteoinductivity - Cell Migration, BMP Production and Signalling Turns a Key.

In this work we dissected the osteoinductive properties of selected, PLGA-based scaffolds enriched with gel-derived bioactive glasses (SBGs) of either binary SiO2-CaO or ternary SiO2-CaO-P2O5 system, differing in CaO/SiO2 ratio (i.e. high -or low-calcium SBGs). To assess the inherent ability of the scaffolds to induce osteogenesis of human bone marrow stromal cells (BMSC), the study was designed to avoid any osteogenic stimuli beyond the putative osteogenic SBG component of the studied scaffolds. The bioactivity and porosity of scaffolds were confirmed by SBF test and porosimetry. Condition media (CM) from BMSC-loaded scaffolds exhibited increased Ca and decreased P content corresponding to SBGs CaO/SiO2 ratio, whereas Si content was relatively stable and overall lower in CM from scaffolds containing binary SBGs. CM from cell-loaded scaffolds containing high-calcium, binary SBGs promoted migration of BMSC and BMP-response in reporter osteoblast cell line. BMSC culture on these scaffolds or the ones containing ternary, low-calcium SBGs resulted in the activation of BMP-related signaling and expression of several osteogenic markers. Ectopic bone formation was induced by scaffolds containing binary SBGs, but high-calcium ones produced significantly more osteoid. Scaffolds containing ternary SBGs negatively influenced the expression of osteogenic transcription factors and Cx43, involved in cell-cell interactions. High-calcium scaffolds stimulated overall higher Cx43 expression. We believe the initial cell-cell communication may be crucial to induce and maintain osteogenesis and high BMP signaling on the studied scaffolds. The presented scaffolds' biological properties may also constitute new helpful markers to predict osteoinductive potential of other bioactive implant materials.

Bone Formation on Murine Cranial Bone by Injectable Cross-Linked Hyaluronic Acid Containing Nano-Hydroxyapatite and Bone Morphogenetic Protein.

New injection-type bone-forming materials are desired in dental implantology. In this study, we added nano-hydroxyapatite (nHAp) and bone morphogenetic protein (BMP) to cross-linkable thiol-modified hyaluronic acid (tHyA) and evaluated its usefulness as an osteoinductive injectable material using an animal model. The sol (ux-tHyA) was changed to a gel (x-tHyA) by mixing with a cross-linker. We prepared two sol−gel (SG) material series, that is, x-tHyA + BMP with and without nHAp (SG I) and x-tHyA + nHAp with and without BMP (SG II). SG I materials in the sol stage were injected into the cranial subcutaneous connective tissues of mice, followed by in vivo gelation, while SG II materials gelled in Teflon rings were surgically placed directly on the cranial bones of rats. The animals were sacrificed 8 weeks after implantation, followed by X-ray analysis and histological examination. The results revealed that bone formation occurred at a high rate (>70%), mainly as ectopic bone in the SG I tests in mouse cranial connective tissues, and largely as bone augmentation in rat cranial bones in the SG II experiments when x-tHyA contained both nHAp and BMP. The prepared x-tHyA + nHAp + BMP SG material can be used as an injection-type osteoinductive bone-forming material. Sub-periosteum injection was expected.

Diffuse Pulmonary Ossification with Connective Tissue Weakness Potentially Due to Vascular Ehlers-Danlos Syndrome.

A 30-year-old non-smoking man was referred to our hospital for the further examination of abnormal shadows revealed by chest X-ray. He had mild shortness of breath. Chest computed tomography revealed a fine-grained dendritic shadow with diffuse calcification in both lungs and as well as emphysematous changes in the upper lung lobes. A surgical lung biopsy histology revealed diffuse pulmonary ossification complicated with lung laceration, vascular disruption, hemosiderosis, and emphysema, suggesting vascular Ehlers-Danlos syndrome (vEDS). However, the patient had no external physical signs or family history of vEDS and no COL3A1 gene mutations. We are closely monitoring this patient in the clinic.

Cell-free, quantitative mineralization measurements as a proxy to identify osteoinductive bone graft substitutes.

Some synthetic bone graft substitutes (BGS) can trigger ectopic bone formation, which is the hallmark of osteoinduction and the most important prerequisite for the repair of large bone defects. Unfortunately, measuring or predicting BGS osteoinductive potential based on in vitro experiments is currently impossible. A recent study claimed that synthetic BGS can induce bone formation ectopically if they create a local homeostatic imbalance during their in vivo mineralization. This raised the hope that a simple cell free in vitro mineralization experiment would correlate with osteoinduction. The aim of the present study was therefore to assess the ability of a quantitative in vitro mineralization test to predict and rank the osteoinductive potential of BGS. Eight calcium phosphate BGS already tested ectopically in 9 different in vivo studies were used for that purpose. The experiment was able to identify materials that are reliably osteoinductive from those that are not, but was inaccurate in ranking the osteoinductive materials between each other. Chemical contaminants (Ca2+, Mg2+, H+, OH-, PO43-) present in some of the BGS affected the in vitro mineralization experiment results, but not in a direction that could explain the different rankings. In conclusion, this study suggests that an in vitro experiment can be used as a fast and reliable screening tool to identify osteoinductive BGS and underline the need to study ionic contaminants on calcium phosphate BGS.

Novel three-dimensional bioglass functionalized gelatin nanofibrous scaffolds for bone regeneration.

The clinical use of FDA-approved bone morphogenetic proteins (BMPs) are impeded by high costs, super-high dosage requirement, short half-life, and other undesirable side effects. Therefore, designing a biomaterial that can promote new bone formation without using exogenous BMPs is highly desirable in clinical applications. In the present work, a new kind of nanofibrous scaffold composed of gelatin and 45S5 bioglass (GF/45S5 BG) was prepared through thermally induced phase separation method together with the particle leach technique (TIPS&P). In addition to the significantly higher mechanical strength, the composite scaffolds (GF/45S5 BG) significantly increased osteogenic differentiation of human mesenchymal stem cells (hMSCs) in vitro compared with the neat scaffold (GF) without adding other biological agents, for example, BMPs or hormones. Most importantly, our in vivo studies also indicated that GF/45S5 BG scaffolds could directly promote ectopic bone regeneration in SD rats without exogenous BMP2. In summary, both in vitro and in vivo results indicated that the novel 45S5 bioglass functionalized GF nanofibrous scaffold is a promising alternative for bone tissue engineering.

The implication of the notch signaling pathway in biphasic calcium phosphate ceramic-induced ectopic bone formation: A preliminary experiment.

Calcium phosphate (BCP) ceramic is a promising material in bone regeneration because it was proved biocompatible, osteoconductive, osteoinductive, and effective. Although it manifests favorable characteristics in critical-sized bone defects repair, the mechanism of its osteoinduction is still unclear. In the present study, we studied the mechanism of ectopic bone formation, with interest in the Notch signaling pathway. BCP ceramics with or without Notch signaling inhibitor RO4929097 were cocultured with bone marrow-derived stem cells in vitro. The expression of osteogenesis (OPN/Col/Runx2) and Notch signaling pathway-related genes (Hes1/Jagged/Notch1) were increased in the BCP group compared with the control group without BCP but significantly decreased after adding RO4929097. Furthermore, a higher level of alkaline phosphatase activity was observed in the BCP group compared with RO4929097 and control group separately. For further confirmation, the intramuscularly ectopic implantation models of Beagle dogs were used. Quantitative real-time polymerase chain reaction showed a similar trend with the in vitro experiment. Histological and histomorphometric analysis indicated that bone formation was delayed by RO4929097. These findings illustrated that the Notch signaling pathway plays a pivotal role in bone formation induced by BCP; Notch signaling pathway may positively influence ectopic bone formation by promoting BMSCs to differentiate toward osteoblasts.

Superior CKIP-1 sensitivity of orofacial bone-derived mesenchymal stem cells in proliferation and osteogenic differentiation compared to long bone-derived mesenchymal stem cells.

Maxillofacial bone defects caused by multiple factors, including congenital deformations and tumors, have become a research focus in the field of oral medicine. Bone tissue engineering is increasingly regarded as a potential approach for maxillofacial bone repair. Mesenchymal stem cells (MSCs) with different origins display various biological characteristics. The aim of the present study was to investigate the effects of casein kinase­2 interaction protein­1 (CKIP­1) on MSCs, including femoral bone marrow­derived MSCs (BMMSCs) and orofacial bone­derived MSCs (OMSCs), isolated from the femoral and orofacial bones of wild­type (WT) and CKIP­1 knockout (KO) mice. MSCs were isolated using collagenase II and the main biological characteristics, including proliferation, apoptosis and osteogenic differentiation, were investigated. Subcutaneous transplantation of MSCs in mice was also performed to assess ectopic bone formation. MTT and clone formation assay results indicated that cell proliferation in the KO group was increased compared with the WT group, and OMSCs exhibited significantly increased levels of proliferation compared with BMMSCs. However, the proportion of apoptotic cells was not significantly different between CKIP­1 KO OMSCs and BMMSCs. Furthermore, it was revealed that osteogenic differentiation was increased in CKIP­1 KO MSCs compared with WT MSCs, particularly in OMSCs. Consistent with the in vitro results, enhanced ectopic bone formation was observed in CKIP­1 KO mice compared with WT mice, particularly in OMSCs compared with BMMSCs. In conclusion, the present results indicated that OMSCs may have a superior sensitivity to CKIP­1 in promoting osteogenesis compared with BMMSCs; therefore, CKIP­1 KO in OMSCs may serve as an efficient strategy for maxillofacial bone repair.

Minimally Invasive Lumbar Decompression and Removal of Symptomatic Heterotopic Bone Formation After Spinal Fusion with Recombinant Human Bone Morphogenetic Protein-2.

We present a case of symptomatic heterotopic bone formation following revision of posterolateral lumbar fusion/instrumentation and "off-label" use of recombinant human bone morphogenetic protein-2, treated successfully with the use of a minimally invasive tubular approach.