Normal Bone Formation Research Articles

Lack of Nuclear Localization of the Creb3l1 Transcription Factor Causes Defects in Caudal Fin Bifurcation in Zebrafish Danio rerio.

The formation of normal bone and bone healing requires the cAMP-responsive element binding protein 3-like-1 (Creb3l1) transmembrane transcription factor, as deletion of the murine CREB3L1 results in osteopenic animals with limited capacity to repair bone after a fracture. Creb3l1 undergoes regulated intramembrane proteolysis (RIP) to release the N-terminal transcription activating (TA) fragment that enters the nucleus and regulates the expression of target genes. To expand our understanding of Creb3l1's role in skeletal development and skeletal patterning, we aimed to generate animals expressing only the TA fragment of Creb3l1 lacking the transmembrane domain and thereby not regulated through RIP. However, the CRISPR/Cas9-mediated genome editing in zebrafish Danio rerio caused a frameshift mutation that added 56 random amino acids at the C-terminus of the TA fragment (TA+), making it unable to enter the nucleus. Thus, TA+ does not regulate transcription, and the creb3l1TA+/TA+ fish do not mediate creb3l1-dependent transcription. We document that the creb3l1TA+/TA+ fish exhibit defects in the patterning of caudal fin lepidotrichia, with significantly distalized points of proximal bifurcation and decreased secondary bifurcations. Moreover, using the caudal fin amputation model, we show that creb3l1TA+/TA+ fish have decreased regeneration and that their regenerates replicate the distalization and bifurcation defects observed in intact fins of creb3l1TA+/TA+ animals. These defects correlate with altered expression of the shha and ptch2 components of the Sonic Hedgehog signaling pathway in creb3l1TA+/TA+ regenerates. Together, our results uncover a previously unknown intersection between Creb3l1 and the Sonic Hedgehog pathway and document a novel role of Creb3l1 in tissue patterning.

Endosteal stem cells at the bone-blood interface: A double-edged sword for rapid bone formation: Bone marrow endosteal stem cells provide a robust source of bone-making osteoblasts both in normal and abnormal bone formation.

Endosteal stem cells are a subclass of bone marrow skeletal stem cell populations that are particularly important for rapid bone formation occurring in growth and regeneration. These stem cells are strategically located near the bone surface in a specialized microenvironment of the endosteal niche. These stem cells are abundant in young stages but eventually depleted and replaced by other stem cell types residing in a non-endosteal perisinusoidal niche. Single-cell molecular profiling and in vivo cell lineage analyses play key roles in discovering endosteal stem cells. Importantly, endosteal stem cells can transform into bone tumor-making cells when deleterious mutations occur in tumor suppressor genes. The emerging hypothesis is that osteoblast-chondrocyte transitional identities confer a special subset of endosteal stromal cells with stem cell-like properties, which may make them susceptible for tumorigenic transformation. Endosteal stem cells are likely to represent an important therapeutic target of bone diseases caused by aberrant bone formation.

Vitamin D deficiency rickets in a toddler.

Nutritional rickets is the failure of normal bone formation in children, caused by vitamin D deficiency, low calcium intake, or a combination of both. In the United States, prolonged breastfeeding without vitamin D supplementation is a major risk factor. Increasing awareness of the rationale for and importance of vitamin D supplements for all breastfed infants and children should reduce the incidence of vitamin D deficiency rickets and prevent bone deformity.

Calcium sensing and signaling are impaired in the lumbar spine of a rat model of congenital kyphosis.

Kyphosis involves spines curving excessively backward beyond their physiological curvature. Although the normal structure of the spinal vertebrae is extremely important for maintaining posture and the normal function of the thoracic and abdominal organs, our knowledge concerning the pathogenesis of the disease is insufficient. We herein report that the downregulation of the calcium signaling pathway is involved in the pathogenesis of congenital kyphosis. The third to fifth lumbar spine segments, the kyphotic region of Ishibashi (IS) rats, which are used as a model of congenital kyphoscoliosis, were collected. A DNA microarray, quantitative PCR, Western blotting, and immunohistochemistry were used to measure the expression of genes and proteins related to intracellular calcium signaling. We found that the expression of calcium-sensing receptor (CaSR) and transient receptor potential vanilloid 1 (Trpv1)-two receptors involved in the calcium signaling-was decreased in the lumbar spine of IS rats. We also observed that the number of CaSR-immunoreactive and Trpv1-immunoreactive cells in the lumbar spine of IS rats was lower than in wild-type rats. Furthermore, the expression of intracellular molecules downstream of these receptors, such as phosphorylated protein kinase C, c-Jun N-terminal kinase, and neural EGFL-like 1, was also reduced. In fact, the calcium content in the lumbar spine of IS rats was significantly lower than that in wild-type rats. These results indicate that adequate calcium signaling is extremely important for the regulation of normal bone formation and may also be a key factor for understanding the pathogenesis of congenital kyphosis.

Dentin Matrix Protein 1 Regulates Mineralization of MC3T3-E1 Cells via the TNAP-ANK-ENPP1 Axis.

Dentin matrix protein 1 (DMP1) is central to matrix mineralization. Clarification of the function of DMP1 is crucial to understanding normal bone formation and pathological calcification. The tissue-nonspecific alkaline phosphatase (TNAP) -progressive ankylosing enzyme (ANK) -extracellular nucleotide pyrophosphatase/phosphodiesterase-1 (ENPP1) axis induces deposition of hydroxyapatite (HA) and pyrophosphate dehydrate (CPPD) by regulating pyrophosphate (PPi). Here, we investigated the mechanism by which DMP1 and the TNAP-ANK-ENPP1 axis participate in mineralization. Expression of DMP1, TNAP, NPP1, and ANK genes in MC3T3-E1 cells was detected by RT-qPCR before and after treatment with DMP1 siRNA. An enzyme-linked immunosorbent assay was used to determine expression of DMP1 protein, TNAP activity was detected by SIGMAFAST p-nitrophenyl phosphate tablets, and mineralization of osteoblasts was determined by alizarin red staining. PPi levels were determined radiometrically and equalized for cell DNA. Levels of calcium, inorganic phosphate, zinc, and magnesium were assessed by standard laboratory techniques. After DMP1 gene silencing, expressions of TNAP, ENPP1, and ANK were correspondingly reduced. DMP1 altered extravesicular and intravesicular ion levels through the TNAP-ENPP1-ANK axis in MC3T3-E1 cells. DMP1 regulated mineralization of MC3T3-E1 cells via the TNAP-ANK-ENPP1 axis and affected TNAP activity by two processes-rapid regulation of the Zn2+ transporter (ZnT) and transcriptional regulation of hysteresis. However, DMP1 may affect expression of ENPP1 and ANK only via hysteresis transcriptional regulation. DMP1, as a calcium trap or catalytic enzyme, appears to have a role in collagen mineralization.

Histological Assessment of Endochondral Ossification and Bone Mineralization

Finely tuned cartilage mineralization, endochondral ossification, and normal bone formation are necessary for normal bone growth. Hypertrophic chondrocytes in the epiphyseal cartilage secrete matrix vesicles, which are small extracellular vesicles initiating mineralization, into the intercolumnar septa but not the transverse partitions of the cartilage columns. Bone-specific blood vessels invade the unmineralized transverse septum, exposing the mineralized cartilage cores. Many osteoblast precursors migrate to the cartilage cores, where they synthesize abundant bone matrices, and mineralize them in a process of matrix vesicle-mediated bone mineralization. Matrix vesicle-mediated mineralization concentrates calcium (Ca) and inorganic phosphates (Pi), which are converted into hydroxyapatite crystals. These crystals grow radially and are eventually get out of the vesicles to form spherical mineralized nodules, leading to collagen mineralization. The influx of Ca and Pi into the matrix vesicle is regulated by several enzymes and transporters such as TNAP, ENPP1, PiT1, PHOSPHO1, annexins, and others. Such matrix vesicle-mediated mineralization is regulated by osteoblastic activities, synchronizing the synthesis of organic bone material. However, osteocytes reportedly regulate peripheral mineralization, e.g., osteocytic osteolysis. The interplay between cartilage mineralization and vascular invasion during endochondral ossification, as well as that of osteoblasts and osteocytes for normal mineralization, appears to be crucial for normal bone growth.

Endothelial-to-osteoblast transition in normal mouse bone development

Metastatic prostate cancer (PCa) in bone induces bone-forming lesions. We have previously shown that PCa-induced bone originates from endothelial cells (ECs) that have undergone EC-to-osteoblast (OSB) transition. Here, we investigated whether EC-to-OSB transition also occurs during normal bone formation. We developed an EC and OSB dual-color reporter mouse (DRM) model that marks EC-OSB hybrid cells with red and green fluorescent proteins. We observed EC-to-OSB transition (RFP and GFP co-expression) in both endochondral and intramembranous bone formation during embryonic development and in adults. Co-expression was confirmed in cells isolated from DRM. Bone marrow- and lung-derived ECs underwent transition to OSBs and mineralization in osteogenic medium. RNA-sequencing revealed GATA family transcription factors were upregulated in EC-OSB hybrid cells and knockdown of GATA3 inhibited BMP4-induced mineralization. Our findings support that EC-to-OSB transition occurs during normal bone development and suggest a new paradigm regarding the endothelial origin of OSBs.

Osificación heterotópica en isquion. Localización infrecuente.

Heterotopic ossifications (HO) are an alteration characterized histologically and radiographically by the formation of normal bony bone in soft tissues where its formation does not normally correspond. The pathology can affect the bones or the joints. It can originate in any extraskeletal location, where undifferentiated mesenchymal cells are found. Multifactorial etiology and cause still completely unknown. They can be found associated with arthroplasty, burns and a wide group of neurological injuries, including spinal cord injury, traumatic brain injury, anoxic brain injuries, cerebrovascular accidents, etc... In patients with central nervous system (CNS) damage, HO is a common complication, ranging from 11% to 76% of cases. In spinal cord injury, the frequency of HO varies from 5% to 60% depending on the study and whether the diagnosis is based on clinical symptoms or standard radiographic results. Symptomatic HO develops in approximately 10% of patients. The most commonly affected joint is the hip. Next in frequency is the knee, with a medial location (54.7%). It can also appear in other places such as the elbow, spine... less frequently. The appearance in the ischium is much less frequent. The indication for surgical treatment will only be in case of mobility restriction, hygiene or clothing limitation and risk of ulceration. This is going to be the case.

Surgical placement of third molars to replace other teeth: “spare wheels”

The prognostic improvement for dental auto-transplants is the result of a better understanding in the physiopathology of the periodontium and also of the refining of the technical operative procedure. This fact allows the use of third molars as donor site for the replacement of absent, or first or second badly decayed molars. This possible therapy is a supplement argument for the conservation of third molars and their extraction must be indicated only when the anatomic situation presents a pathological risk. If, in adults, it is possible to compare the advantages and disadvantages with the classical implant or prosthetic techniques, it should however be considered as first choice therapy for adolescents and young adults. Indeed, in these cases, the lower risk of root ankylosis avoids passive intrusion and the osteogenic effect of the desmodontium allows normal alveolar bone formation.

Analysis of Chemotactic Property of CCN2/CTGF in Intramembranous Osteogenesis.

Chemotaxis is a directed migration of cells in response to a gradient of extracellular molecules called chemoattractants. Development, growth, remodeling, and fracture healing of bones are advanced through intramembranous osteogenesis. Chemotaxis of preosteoblasts toward future bone formation sites observed in the early stage of intramembranous osteogenesis is a critical cellular process for normal bone formation. However, molecular biological mechanisms of the chemotaxis of preosteoblasts are not fully understood. We have recently clarified, for the first time, the critical role of the cellular communication network factor 2 (CCN2)/connective tissue growth factor (CTGF)-integrin α5-Ras axis for chemotaxis of preosteoblasts during new bone formation through intramembranous osteogenesis. In this chapter, we describe in detail the procedures of the in vivo and in vitro assays to investigate the chemotactic property of CCN2/CTGF and its underlying molecular biological mechanisms during intramembranous osteogenesis.

Osseous choristoma: Report of two cases in oral cavity

Osseous choristoma is the formation of normal mature bone at an abnormal position. These are rare benign tumor-like growth usually asymptomatic except growth or swelling as the clinical presentation. In oral cavity, osseous choristoma is rare and commonly found in posterior third of the tongue. Clinically it is misdiagnosed as malignancy because of its hardness. Clinically, it is also mistaken for other oral cavity lesions, especially those of minor salivary gland lesions of oral cavity. Hence, awareness amongst clinicians, general practitioners and oral pathologists about oral osseous choristomas is necessary. Here, we report two cases of osseous choristoma in the oral cavity located at sites other than tongue.

Fibrous dysplasia of the paranasal sinuses: case report

<p class="abstract">Fibrous dysplasia (FD) is a rare congenital non-malignant bone tumor that affects the skeletal system development and interferes with normal bone formation. It’s non-hereditary and its exact cause remains unknown. The condition is characterized by fibro-osseous connective tissue gradually replacing normal bone and marrow leading to the formation of bone that’s fragile and prone to fractures. It is not uncommon for the disease to affect craniofacial bones but the sinonasal cavity involvement remains extremely rare. We presented a case of an 80-years-old male patient who reported nasal obstruction and a long-term swelling in the upper jaw that dates back over 20 years resulting in facial deformities and unilateral proptosis from compression exerted on the eye by the tumor. The patient reported having taken drugs to treat symptoms especially nasal congestion. But his dyspnea persisted. Physical examination, computed tomography (CT) scan and MRI have been elaborated and discussed. And on last resort, surgery was recommended.</p>

The Emerging Role of Cell Transdifferentiation in Skeletal Development and Diseases.

The vertebrate musculoskeletal system is known to be formed by mesenchymal stem cells condensing into tissue elements, which then differentiate into cartilage, bone, tendon/ligament, and muscle cells. These lineage-committed cells mature into end-stage differentiated cells, like hypertrophic chondrocytes and osteocytes, which are expected to expire and to be replaced by newly differentiated cells arising from the same lineage pathway. However, there is emerging evidence of the role of cell transdifferentiation in bone development and disease. Although the concept of cell transdifferentiation is not new, a breakthrough in cell lineage tracing allowed scientists to trace cell fates in vivo. Using this powerful tool, new theories have been established: (1) hypertrophic chondrocytes can transdifferentiate into bone cells during endochondral bone formation, fracture repair, and some bone diseases, and (2) tendon cells, beyond their conventional role in joint movement, directly participate in normal bone and cartilage formation, and ectopic ossification. The goal of this review is to obtain a better understanding of the key roles of cell transdifferentiation in skeletal development and diseases. We will first review the transdifferentiation of chondrocytes to bone cells during endochondral bone formation. Specifically, we will include the history of the debate on the fate of chondrocytes during bone formation, the key findings obtained in recent years on the critical factors and molecules that regulate this cell fate change, and the role of chondrocyte transdifferentiation in skeletal trauma and diseases. In addition, we will also summarize the latest discoveries on the novel roles of tendon cells and adipocytes on skeletal formation and diseases.

Major approaches and clinical outcomes of bone regeneration for dental implants: a systematic review

Introduction: In the last decades, the number of dental implant procedures has increased in the world, with about 1.0 million dental implants per year. When a tooth is lost in the posterior region of the maxilla, there is a natural resorption of the alveolar process. There are several surgical techniques that can be used to reconstruct the atrophic alveolar ridge. Objective: A systematic review was carried out to present the key considerations of bone regeneration and bone, cellular and molecular grafts for adequate bone formation for successful dental implants. Methods: The present study followed a systematic review model, following the rules of systematic review – PRISMA. The search strategy was performed in the PubMed, Cochrane Library, Web of Science and Scopus, and Google Scholar databases. The quality of the studies was based on the GRADE instrument and the risk of bias was analyzed according to the Cochrane instrument. Results and Conclusion: The total of 132 articles were found. A total of 74 articles were fully evaluated and 31 were included in this study. Normal bone formation and tissue repair involve coordinated interaction between bone-forming cells and biological signals. TNF-α stimulates bone and cartilage resorption and inhibits collagen and proteoglycan synthesis. IL-1 induces the expression of a wide variety of cytokines. LIF and IL-6 are two such molecules that are known to stimulate the differentiation of mesenchymal progenitor cells into the osteoblastic lineage. The bioactivation of the dental implant surface with FRP has been described and discussed by the scientific community as a surface treatment for the stimulation and acceleration of the osseointegration process, as well as to achieve greater primary stability to the implant. The combination of FRP and Bio-Oss® has been studied with good clinical results, reducing healing time from 180 days to approximately 106 days. The use of bone grafts significantly improves the residual alveolar ridge regardless of the membrane used. Furthermore, implants placed in fresh extraction sockets with and without elevation of the mucoperiosteal flap can be successfully performed with augmentation procedures.

Gli1+ Osteogenic Progenitors Contribute to Condylar Development and Fracture Repair

The condyle plays a pivotal role in mandible development, which is regulated by various signaling molecules. The hedgehog (Hh) signaling pathway is known to modulate several processes during bone formation. However, the role of Gli1, as the read-out of Hh signaling activity, in condylar development and fracture healing has not been clarified. In this study, we discovered that a population of Gli1+ cells residing immediately below the cartilage functions as osteogenic progenitors by using Gli1-Cre ERT2 ;tdTomato mice. These Gli1+ cells contributed to nearly all osteoblasts in the subchondral bone during condyle postnatal development. Interestingly, Gli1-lineage cells could differentiate into osteoblasts and chondrocytes during fracture healing. Inhibiting Wnt/β-catenin signaling downregulated the proliferation and differentiation of Gli1+ cells in vitro. These findings suggest that Gli1+ progenitor cells participate in not only normal bone formation but also fracture healing; moreover, these cells may provide a potential target for promoting bone regeneration of the mandible.

Effect of platelet-rich plasma on bone regenerate consolidation in distraction osteogenesis: An experimental study in rabbits.

The aim of this study was to determine the effect of platelet-rich plasma (PRP) on bone regenerate consolidation in a rabbit model of distraction osteogenesis. In this study, 12 male New Zealand rabbits weighing 1600 to 2000 g were used. All the rabbits were randomly divided into two groups (n = 6 per group): PRP group and control group. A two-ring, circular external fixator was applied to the right tibia of each rabbit in both groups. After corticotomy, all the tibiae were distracted at a rate of 0.5 mm/day for 20 days. PRP was injected to the osteotomy sites on the 7th, 14th, and 21st days postoperatively. Mineral density of the new bone tissue formed in the distraction zone was measured using quantitative computed tomography in the 3rd, 4th, and 5th weeks. At the end of the 6th week, the animals were sacrificed, and the specimens were evaluated biomechanically and histologically. Microcomputed tomography assessment showed significant bone mineral density increase from the 3rd to the 6th week (62.3% and 43.7% for the PRP and control groups, respectively). In the PRP group, the measurements on the 3rd, 4th, and 5th weeks were 416 ± 29, 487 ± 9.9, and 675 ± 37.8 HU (Hounsfield units), respectively, whereas in the control group were 313, 374, and 450 HU, respectively. In the comparison of weekly measurements of the two groups, the increase in bone density in the PRP group was higher than that in the control group (P < 0.001). During the mechanical tests, in the PRP group, the mean torsion was 46.50°and the mean torque 0.53 Nm, while in the control group, the mean torsion was 19.33°and the mean torque 0.65 Nm. The mechanical analysis of the groups revealed significant differences in the mean maximum torsion angles (P = 0.024). The histological examination showed that both groups had external and internal periosteal calli. Callus tissue in four rabbits in the PRP group and two rabbits in the control group was remodeled; normal bone formation occurred and distracted bone ends were completely healed. The results of the present study have indicated that PRP injection can enhance bone regenerate consolidation and increase bone mineral density during the healing process of distraction osteogenesis.

Osteoma in the Lumbar Spinal Canal as a Rare Cause of Sciatica: A Case Report and Literature Review

Among the etiological causes of lumbar pain, the osteoma is a rare benign bone tumor of the spine. The osteoma is a slowly growing benign tumor arising from normal bone formation in the periosteum. Osteomas are usually seen in the skull and long bones, but in the literature, rare cases of osteomas affecting the vertebra corpus, pedicle and posterior element are encountered. In this case, a 28-year-old man presented with an osteoma originating from the left L5-S1 lateral recess and causing lumbo-crural sciatica. The osteoma originating from lateral foramina causes lateral spinal stenosis, sciatica and neuropathic pain in the patient. On magnetic resonance imaging, osteoma was not detected and the diagnosis was confirmed by computed tomography. At the same time, when the patient's anamnesis was deepened, accompanying spondyloarthropathy was diagnosed.

MiR-452-3p inhibited osteoblast differentiation by targeting Smad4

Osteoblast differentiation is a complex process that is essential for normal bone formation. A growing number of studies have shown that microRNAs (miRNAs) are key regulators in a variety of physiological and pathological processes, including osteogenesis. In this study, BMP2 was used to induce MC3T3-E1 cells to construct osteoblast differentiation cell model. Then, we investigated the effect of miR-452-3p on osteoblast differentiation and the related molecular mechanism by RT-PCR analysis, Western blot analysis, ALP activity, and Alizarin Red Staining. We found that miR-452-3p was significantly downregulated in osteoblast differentiation. Overexpression miR-452-3p (miR-452-3p mimic) significantly inhibited the expression of osteoblast marker genes RUNX2, osteopontin (OPN), and collagen type 1 a1 chain (Col1A1), and decreased the number of calcium nodules and ALP activity. In contrast, knockdown miR-452-3p (miR-452-3p inhibitor) produced the opposite effect. In terms of mechanism, we found that Smad4 may be the target of miR-452-3p, and knockdown Smad4 (si-Smad4) partially inhibited the osteoblast differentiation enhanced by miR-452-3p. Our results suggested that miR-452-3p plays an important role in osteoblast differentiation by targeting Smad4. Therefore, miR-452-3p is expected to be used in the treatment of bone formation and regeneration.

Increased Potential of Bone Formation with the Intravenous Injection of a Parathyroid Hormone-Related Protein Minicircle DNA Vector.

Osteoporosis is commonly treated via the long-term usage of anti-osteoporotic agents; however, poor drug compliance and undesirable side effects limit their treatment efficacy. The parathyroid hormone-related protein (PTHrP) is essential for normal bone formation and remodeling; thus, may be used as an anti-osteoporotic agent. Here, we developed a platform for the delivery of a single peptide composed of two regions of the PTHrP protein (1–34 and 107–139); mcPTHrP 1–34+107–139 using a minicircle vector. We also transfected mcPTHrP 1–34+107–139 into human mesenchymal stem cells (MSCs) and generated Thru 1–34+107–139-producing engineered MSCs (eMSCs) as an alternative delivery system. Osteoporosis was induced in 12-week-old C57BL/6 female mice via ovariectomy. The ovariectomized (OVX) mice were then treated with the two systems; (1) mcPTHrP 1–34+107–139 was intravenously administered three times (once per week); (2) eMSCs were intraperitoneally administered twice (on weeks four and six). Compared with the control OVX mice, the mcPTHrP 1–34+107–139-treated group showed better trabecular bone structure quality, increased bone formation, and decreased bone resorption. Similar results were observed in the eMSCs-treated OVX mice. Altogether, these results provide experimental evidence to support the potential of delivering PTHrP 1–34+107–139 using the minicircle technology for the treatment of osteoporosis.

Gli1+ progenitors mediate bone anabolic function of teriparatide via Hh and Igf signaling.

SUMMARYTeriparatide is the most widely prescribed bone anabolic drug in the world, but its cellular targets remain incompletely defined. The Gli1+ metaphyseal mesenchymal progenitors (MMPs) are a main source for osteoblasts in postnatal growing mice, but their potential response to teriparatide is unknown. Here, by lineage tracing, we show that teriparatide stimulates both proliferation and osteoblast differentiation of MMPs. Single-cell RNA sequencing reveals heterogeneity among MMPs, including an unexpected chondrocyte-like osteoprogenitor (COP). COP expresses the highest level of Hedgehog (Hh) target genes and the insulin-like growth factor 1 receptor (Igf1r) among all cell clusters. COP also expresses Pth1r and further upregulates Igf1r upon teriparatide treatment. Inhibition of Hh signaling or deletion of Igf1r from MMPs diminishes the proliferative and osteogenic effects of teriparatide. The study therefore identifies COP as a teriparatide target wherein Hh and insulin-like growth factor (Igf) signaling are critical for the osteoanabolic response in growing mice.