Human Osteogenesis Research Articles

An oscillating mechanism in the synthesis of doped nanocrystalline calcium carbonate phosphates

A mechanism of the synthesis reactions of doped nanocrystalline calcium carbonate phosphates is considered, and an oscillating type of model is proposed for these reactions. The system is oscillatory in nature and can be represented as a brusselator of the simplest implementation of cubic nonlinearity. Biomaterial obtaining highly active resorption and the transdermal ability to activate the processes of osteogenesis in humans of all ages for the restoration of bone tissue and for strengthening tooth tissue is developed.

Immunohistochemical evidence of lactoferrin in human embryo–fetal bone and cartilage tissues

Lf (lactoferrin) is an 80-kDa iron-binding protein, which has been suggested to promote bone growth in murine models. In view of this, we aimed to analyse the immunohistochemical distribution of Lf in human embryonal and fetal bone and cartilaginous tissues at different gestational weeks in order to evaluate whether a role for this protein might be proposed also in human osteogenesis. Bone and cartilaginous specimens were taken at autopsy from 25 fetuses (8-34 weeks of gestation). Ten samples of human adult bone and cartilage were also submitted to the immunohistochemical procedures. Sections, 4-microm thick, were cut from formalin-fixed paraffin-embedded tissue blocks and stained with a monoclonal antibody against human Lf, following antigen retrieval procedures. Lf immunoreactivity was mainly localized in the mesenchymal cells forming the periosteum as well as in chondroblasts at the eighth gestational week; a strong Lf immunoexpression in immature osteocytes and osteoblasts was noted up to the 18th gestation week, with a considerable decrease by the 24th week. No Lf expression was found in any bone area after the 30th and up to the 34th week of gestation. Our data seem to suggest an important role for Lf as a bone growth regulator in the early phases of the human endochondral ossification, with an anabolic action similar to that previously reported in cell culture lines and in animal models.

MicroRNA hsa-miR-135b Regulates Mineralization in Osteogenic Differentiation of Human Unrestricted Somatic Stem Cells

Unrestricted somatic stem cells (USSCs) have been recently identified in human umbilical cord blood and have been shown to differentiate into lineages representing all 3 germ layers. To characterize microRNAs that may regulate osteogenic differentiation of USSCs, we carried out expression analysis for 157 microRNAs using quantitative RT-PCR before and after osteogenic induction (t = 0.5, 24, 72, 168, 216 h). Three microRNAs, hsa-miR-135b, hsa-miR-224, and hsa-miR-31, were consistently down-regulated during osteogenesis of USSC line 1. Hsa-miR-135b was shown to be the most profoundly down-regulated in osteogenesis of USSC line 1 and further confirmed to be down-regulated in the osteogenic differentiation of 2 additional USSC lines. Function of hsa-miR-135b in osteogenesis of USSCs was examined by retroviral overexpression, which resulted in an evident decreased mineralization, indicating that hsa-miR-135b down-regulation is functionally important for full osteogenic differentiation of USSCs. MicroRNAs have been shown to regulate negatively expression of their target gene(s). To identify putative targets of hsa-miR-135b, we performed cDNA microarray expression analysis. We selected in total 10 transcripts that were down-regulated (>or=2-fold) in response to hsa-miR-135b overexpression at day 7 and day 9 of osteogenic differentiation. The function of most of these targets in human osteogenesis is unknown and requires further investigation. Markedly, quantitative RT-PCR data showed decreased expression of osteogenic markers IBSP and Osterix, both known to be involved in bone mineralization, in osteogenesis of USSCs that overexpress hsa-miR-135b. This finding suggests that hsa-miR-135b may control osteoblastic differentiation of USSCs by regulating expression of bone-related genes.

Flow perfusion bioreactors enhance human osteogenesis in a collagen based biomaterial

Flow perfusion bioreactors enhance human osteogenesis in a collagen based biomaterial

A novel collagen scaffold supports human osteogenesis—applications for bone tissue engineering

Collagen glycosaminoglycan (CG) scaffolds have been clinically approved as an application for skin regeneration. The goal of this study has been to examine whether a CG scaffold is a suitable biomaterial for generating human bone tissue. Specifically, we have asked the following questions: (1) can the scaffold support human osteoblast growth and differentiation and (2) how might recombinant human transforming growth factor-beta (TGF-beta(1)) enhance long-term in vitro bone formation? We show human osteoblast attachment, infiltration and uniform distribution throughout the construct, reaching the centre within 14 days of seeding. We have identified the fully differentiated osteoblast phenotype categorised by the temporal expression of alkaline phosphatase, collagen type 1, osteonectin, bone sialo protein, biglycan and osteocalcin. Mineralised bone formation has been identified at 35 days post-seeding by using von Kossa and Alizarin S Red staining. Both gene expression and mineral staining suggest the benefit of introducing an initial high treatment of TGF-beta(1) (10 ng/ml) followed by a low continuous treatment (0.2 ng/ml) to enhance human osteogenesis on the scaffold. Osteogenesis coincides with a reduction in scaffold size and shape (up to 70% that of original). A notable finding is core degradation at the centre of the tissue-engineered construct after 49 days of culture. This is not observed at earlier time points. Therefore, a maximum of 35 days in culture is appropriate for in vitro studies of these scaffolds. We conclude that the CG scaffold shows excellent potential as a biomaterial for human bone tissue engineering.

Osteogenic-related gene expression profiles of human dental follicle cells induced by dexamethasone1

Human dental follicle cells (hDFC) have the ability to differentiate into mineralized tissue-forming cells during root and periodontal development or osteogenic induction in vitro. The present study aimed to validate the osteogenic induction of hDFC by dexamethasone (DEX) and to explore the changes of related genes responsible for the osteogenic differentiation process. Passage-cultured hDFC were induced by DEX and analyzed for mineralization activity by morphological observation, alkaline phosphatase (ALP) activity, and alizarin red S staining. GEArray Q series human osteogenesis gene array was used to describe large-scale gene expression in treated hDFC compared to the control group. Quantitative real-time RT-PCR was performed to confirm the microarray data by analyzing the expression of 7 critical transcripts. Osteogenic differentiation of hDFC was confirmed by morphological change, elevated ALP activity and calcified nodules. In 96 genes investigated through the microarray analysis, 20 genes were upregulated and 8 genes were downregulated more than 2-fold. The results of the real-time RT-PCR correlated with the microarray analysis. The expression of the transforming growth factor-beta superfamily showed varying degrees of increase, and fibroblast growth factors exhibited a differential changing trend of expression. The expression of most types of collagen genes representative of extracellular matrixes increased under DEX treatment while small mothers against decapentaplegic 6 and 7 expressions significantly decreased. Our results demonstrated that hDFC displayed osteoblastic features in both phenotypic and genotypic traits induced by DEX in vitro.

Fates and osteogenic differentiation potential of human mesenchymal stem cells in immunocompromised mice

Human mesenchymal stem cells (hMSCs) from bone marrow were genetically marked by using a murine leukaemia virus construct encoding enhanced green fluorescent protein (eGFP). The marked cells were either directly implanted into the tibialis anterior muscle or introduced into a variety of other tissue sites in immunocompromised mice (NOD/SCID and C.B-17 SCID/beige) to investigate their fates and differentiation potentials. It was observed that the hMSCs survived for up to 12 weeks and showed site-specific morphological phenotypes. hMSCs delivered by intravenous injection were found mainly in the lungs and were detected rarely in other organs. Histomorphometry showed that, after implantation of hMSCs into the tibialis anterior muscle juxtaskeletally, the areas of reactive host callus formation at 1 and 2 weeks and of ectopic human bone formation at 1 week were significantly increased compared with the control group. Expression of eGFP and human RUNX2, alkaline phosphatase, osteocalcin, osteopontin, and collagen type I mRNAs were detected in mice implanted with the labelled hMSCs but not in sham-treated samples. Active clearance of the reactive callus and ectopic calcified tissue by osteoclast-like tartrate-resistant acid phosphatase-positive cells was observed. We conclude that the eGFP-labelled hMSCs can survive and retain the potential to differentiate morphologically into a variety of apparent mesenchymal phenotypes in vivo. Absolute confirmation of differentiation capacity requires further study and is complicated by known possibilities of fusion of donor and host cells or limited transfer of genetic material. Nevertheless, the genetically marked hMSCs are shown to participate extensively in bone formation and turnover. Control of the host osteoclast/macrophage responses resulting in clearance of formed osteogenic tissue warrants further investigation to promote prolonged human osteogenesis in immunocompromised mice. Furthermore, any proposed general cytotherapeutic strategy for enhanced osteogenesis is likely to require supplementation of local bone-forming biological signals.

Composite Ceramic Bone Graft Substitute in the Treatment of Locally Aggressive Benign Bone Tumours

To report the use of a composite ceramic bone graft substitute containing calcium sulphate and hydroxyapatite (HA) in the treatment of large expansive osteolytic benign bone tumours. 4 women and 9 men aged 8 to 49 (mean, 22) years with aneurysmal bone cysts (n=6) or giant cell tumours (n=7) in the epi- or meta-physeal areas of the lower limbs underwent curettage, phenolisation, and filling with bone graft substitute containing calcium sulphate and HA. The mean tumour size was 38.5 (range, 18-65) ml. The patients were followed up for a mean of 41 (range, 33-52) months. Range of movement, Musculoskeletal Tumor Society Rating Score (MTSRS), and haematological and blood biochemical parameters were measured. Two patients had recurrence at 7 and 9 months, both progressed to grade-III giant cell tumours. One underwent revision with an iliac crest autograft, whereas the other underwent en bloc excision and prosthetic replacement. The 11 other lesions displayed clinical and radiological consolidation at a mean of 4.6 (range, 3-7) months. No restriction of range of movement was observed, except in the patient undergoing prosthetic replacement. The mean MTSRS was 96% (range, 83-100%) of that expected for normal function. During the follow-up period, haematological and blood biochemical parameters stayed within normal limits. Composite bioceramic osteoconductive grafts, which combine porous HA with calcium sulphate, provide a framework for human osteogenesis and avoid donor-site morbidity (autologous bone graft harvesting). Tumour recurrence remains a major concern especially in young patients, as revision invariably requires removal of additional bone, potentially compromising joint integrity.

A type I collagen defect leads to rapidly progressive osteoarthritis in a mouse model

This study was undertaken to test the hypothesis that abnormalities of the subchondral bone can result in osteoarthritis (OA). We used a knockin model of human osteogenesis imperfecta, the Brittle IV (Brtl) mouse, in which defective type I collagen is expressed in bone. OA in individual mice was documented by micro-magnetic resonance imaging (micro-MRI) and micro-computed tomography (micro-CT). Alterations in the knee joints were confirmed by histopathologic and immunohistochemical analysis. In addition, atomic force microscopy (AFM) was used to assess the ultrastructure of the articular cartilage and subchondral bone matrix. Brtl mice had decreased integrity of bone but initially normal articular cartilage. However, by the second month of life, Brtl mice developed alterations of the cartilage that were characteristic of OA, as documented by micro-CT, micro-MRI, and histologic evaluation. In addition, chondrocyte loss and breakdown of the collagen matrix in the residual cartilage were demonstrated using AFM. The Brtl mouse model demonstrates that progressive destruction of articular cartilage characteristic of OA may be secondary to altered architecture of the underlying subchondral bone.

Effect of cyclic-tension force on the expression of osteogenesis genes in human periodontal ligament cells

To investigate the effect of cyclic-tension force on the expression of osteogenesis genes in human periodontal ligament cells (HPDLC). HPDLC were cultured on flexible-bottomed plates and subjected to 12% elongation by strain unit at 6 cycles/min (i.e.5-s elongation and 5-s relaxation) for 48 hours in the experimental groups. GEArray Q series Human Osteogenesis Gene Array was used to identify the genes expressed in HPDLC, including growth factors and associated molecules, extracellular matrix and its associated proteins, cell adhesion molecules and housekeeping genes. The changes in the expression of 96 representative transcripts were determined by arrayed cDNA hybridization. After application of tension force, 21 genes were significantly upregulated, including 10 growth factors and associated molecules, 10 extracellular matrix and its associated protein and 1 cell adhesion molecules. Two genes were significantly downregulated, including 1 growth factors and associated molecules and 1 cell adhesion molecules. The HPDLC can differentiate into osteoblast-like cells by mechanical stretch induction through the activation of some osteogenesis genes.

P28 Effect of chitosan on in vitro human bone marrow stromal cell osteogenesis and angiogenic factor secretion

Effect of chitosan on in vitro human bone marrow stromal cell osteogenesis and angiogenic factor secretion M.H. Pham, H. El-Gabalawy, G. Lavalee, N. Tran-Khanh, M.D. McKee, M. Centola, C.D. Hoemann; Dept Chemical Engineering, Ecole Polytechnique, Montreal, Canada, Division Of Rheumatology, University of Manitoba, Winnipeg, Canada, Institute Of Biomedical Engineering, Ecole Polytechnique, Montreal, Canada, Dentistry Room M, McGil University, Montreal, Canada, Arthritis And Immunology Program, Oklahoma Medical Research Foundation, Oklahoma City, OK, United States of America

Second harmonic generation imaging microscopy studies of osteogenesis imperfecta

We have used quantitative second harmonic generation (SHG) imaging microscopy to investigate the collagen matrix organization in the oim mouse model for human osteogenesis imperfecta (OI). OI is a heritable disease in which the type I collagen fibrils are either abnormally organized or small, resulting in a clinical presentation of recurrent bone fractures and other pathologies related to collagen-comprised tissues. Exploiting the exquisite sensitivity of SHG to supramolecular assembly, we investigated whether this approach can be utilized to differentiate normal and oim tissues. By comparing SHG intensity, fibrillar morphology, polarization anisotropy, and signal directionality, we show that statistically different results are obtained for the wild type (WT) and disease states in bone, tendon, and skin. All these optical signatures are consistent with the collagen matrix in the oim tissues being more disordered, and these results are further consistent with the known weaker mechanical properties of the oim mouse. While the current work shows the ability of SHG to differentiate normal and diseased states in a mouse model, we suggest that our results provide a framework for using SHG as a clinical diagnostic tool for human OI. We further suggest that the SHG metrics described could be applied to other connective tissue disorders that are characterized by abnormal collagen assembly.

Growth factor expression following clinical mandibular distraction osteogenesis in humans and its comparison with existing animal studies

Lengthening the mandible by distraction osteogenesis (DO) is nowadays a well recognized technique in maxillofacial surgery. In this study growth factor expression profiles were examined in biopsies taken from six patients undergoing mandibular DO and compared with findings from a sheep model for mandibular DO. In all patients (and sheep), the ascending ramus was distracted 10-15 mm at a rate of 1mm/day using an intraoral device. Biopsies were taken from the centre of the distraction zone 21 days after completion of distraction. Using standard immunohistochemical techniques, samples were stained for platelet-derived growth factor (PDGF), transforming growth factor beta (TGF-beta), basic fibroblast growth factor (bFGF) and bone morphogenetic proteins-2, -4 and -7 (BMP-2, -4, -7), matrix metalloproteinases-1 and -3 (MMP-1, -3), the vascular endothelial growth factor (VEGF), a marker for endothelial cells (CD-31) and type IV collagen (Col IV). Positive staining for PDGF, bFGF, TGF-beta, BMP-2, -4, and -7 was noted in cells and matrix components. There was intense staining for MMP-1. Strong staining for CD-31 and COL IV was observed adjacent to vessels. VEGF staining was less specific. Similar findings were noted in the sheep model. Growth factor expression in the human distraction site is similar to that in the sheep model.

Expression pattern of the chromosome 21 transcription factor Ets2 in cell‐seeded three‐dimensional bone constructs

The ability to generate new bone for reconstructive surgery use is a major clinical need. Tissue engineering with osteoprogenitor cells isolated from the patient's periosteum and seeded into bioresorbable scaffolds offers a promising approach to the generation of skeletal tissue. To our knowledge, there is no description about the expression of Ets2 in tissue engineered "bone neotissue." The aim of our study was to manufacture cell-seeded three-dimensional bone constructs with human periosteal cells on poly (lactic-co-glycolic acid) polymer fleeces to describe the expression pattern of Ets2 and its target genes osteocalcin and osteopontin; expression analysis of type I collagen, core-binding factor-1, alkaline phosphatase, and osteonectin; the ability of matrix mineralization and ALP enzymatic activity showed the osteogenic character of the constructs. A significant correlation between the expression of Ets2 and osteopontin mRNA (r = -0.70; p < 0.05) could be shown. A 1.35-fold increase of Ets2 expression from days 1 to 9 was detected, followed by a slight decrease from days 11 to 15. Until the end of the culture period, the expression of Ets2 reached a comparable high level as detected on day 9. In contrast, the expression level of osteopontin mRNA reached a maximum at day 7, followed by a progressive 3.04-fold decrease until day 21. This study shows for the first time that Ets2 gene and its transcriptional target genes are expressed in tissue-engineered bone constructs. These findings have the potential to provide much-needed information about the role and function of Ets2 in human osteogenesis processes and creation of "bone neotissue."

BMP and bone formation

Bone Morphogenetic Proteins (BMPs) are secreted proteins that represent a group of the transforming growth factor-beta. BMPs were identified as molecules that induce ectopic bone formation in muscles of mice. The homo- or heterodimetric BMP proteins, which bind to serine/threonine protein-kinase receptor and activate smad and p38MAPK signal transductions, function as potent osteogenic factors. In vivo, it is not clear which BMPs play crucial roles in regulating osteogenesis. Clinical uses of rhBMP-2 and rhBMP-7 have been performed, but we need numerous rhBMPs, which is very expensive, to induce osteogenesis in humans.

Intermittent parathyroid hormone (1–34) enhances mechanical strength and density of new bone after distraction osteogenesis in rats

Distraction osteogenesis is used both for leg lengthening and for bone transportation in the treatment of fractures and nonunions. The main problem with this method is that the time until full recovery may be up to a year, partly because of the time needed for the new formed bone to consolidate and become strong enough for weight bearing. We have studied whether intermittent parathyroid hormone (PTH(1–34)) could accelerate the consolidation of new formed bone after distraction osteogenesis in rats. Forty-seven, 3-months-old male Sprague–Dawley rats underwent lengthening of the right femur using an external fixator. After a middiaphyseal osteotomy and a 7-day latency period, the callus was distracted during 10 days, with a distraction rate of 0.25 mm twice a day. The consolidation time was either 20 days or 40 days after distraction was completed. A dose of 60 μg of human PTH(1–34)/kg body weight/injection or vehicle was given every second day beginning 30 days before the rats were killed. Both femura of each rat were subjected to mechanical testing and dual-energy X-ray absorptiometry. Blinded histological examination was done for the distracted femura. In the 20 days consolidation experiment, PTH(1–34) increased ultimate load (56%), stiffness (117%), total regenerate callus volume (58%), callus BMC (24%) and histologic bone density (35%) compared to untreated distraction osteogenesis specimens. In the 40 days consolidation experiment, PTH(1–34) increased ultimate load (54%), stiffness (55%), callus BMC (33%) and histologic bone density (23%) compared to untreated distraction osteogenesis specimens. Total regenerate callus volume was unchanged. The contralateral femur also became stronger, stiffer and denser under PTH(1–34) treatment, but to a lesser degree. PTH(1–34) might become useful to shorten the consolidation time after distraction osteogenesis in humans.

Radiographic analysis of zebrafish skeletal defects

Systematic identification of skeletal dysplasias in model vertebrates provides insight into the pathogenesis of human skeletal disorders and can aid in the identification of orthologous human genes. We are undertaking a mutagenesis screen for skeletal dysplasias in adult zebrafish, using radiography to detect abnormalities in skeletal anatomy and bone morphology. We have isolated chihuahua, a dominant mutation causing a general defect in bone growth. Heterozygous chihuahua fish have phenotypic similarities to human osteogenesis imperfecta, a skeletal dysplasia caused by mutations in the type I collagen genes. Mapping and molecular characterization of the chihuahua mutation indicates that the defect resides in the gene encoding the collagen I(α1) chain. Thus, chihuahua accurately models osteogenesis imperfecta at the biologic and molecular levels, and will prove an important resource for studies on the disease pathophysiology. Radiography is a practical screening tool to detect subtle skeletal abnormalities in the adult zebrafish. The identification of chihuahua demonstrates that mutant phenotypes analogous to human skeletal dysplasias will be discovered.

BMP Responsiveness in Human Mesenchymal Stem Cells

Bone morphogenetic proteins (BMPs) are well known to induce bone formation in animal models and can promote osteogenesis in cultures of multipotential mesenchymal stem cells (MSC) isolated from rat and mouse bone marrow. However, clinical trials of BMPs suggest that BMPs are relatively ineffective inducers of osteogenesis in humans. Recent studies from our lab indicate that when human bone marrow MSC are placed in primary culture, osteogenesis can be induced by dexamethasone (Dex), but not by BMP-2, -4, or -7. We have therefore investigated components of BMP signaling pathways in human MSC. First passage cells, derived from the bone marrow of patients undergoing hip replacement surgery, were cultured with ascorbate phosphate and treated with 100 nM dexamethasone (Dex), 100 ng/ml BMP, or both. After 6 days, alkaline phosphatase activity of cell extracts was measured, and RNA was extracted for RT-PCR analysis of mRNA levels. Among human MSC samples from more than a dozen patients, only one patient sample showed significantly elevated alkaline phosphatase after exposure to BMP; the rest responded to Dex but not BMP. Analysis of mRNA from cultured human MSC indicated that, while Dex treatment caused increased levels of mRNA for alkaline phosphatase, BMP did not. Noggin is a BMP-binding protein that is upregulated by BMPs. BMP-treated human MSC cultures that did not show increased alkaline phosphatase did express elevated levels of noggin mRNA, indicating that the cells are capable of some BMP response. Our results suggest that BMP signaling in mesenchymal stem cells utilizes more than one system for transcriptional activation. The inability of most human MSC to activate transcription of the alkaline phosphatase gene implies that a defect exists in the system required for induction of the osteoblast phenotype.

Leukaemia inhibitory factor (lif) is expressed in hypertrophic chondrocytes and vascular sprouts during osteogenesis.

Avascular cartilage is replaced by highly vascularized bone tissue during endochondral ossification, a process involving capillary invasion of calcified hypertrophic cartilage in association with apoptosis of hypertrophic chondrocytes, degradation of cartilage matrix and deposition of bone matrix. All of these events are closely controlled, especially by cytokines and growth factors. Leukaemia inhibitory factor (LIF), a member of the gp130 cytokine family, is involved in osteoarticular tissue metabolism and might participate in osteogenesis. Immunohistochemical staining showed that LIF is expressed in hypertrophic chondrocytes and vascular sprouts of cartilage and bone during rat and human osteogenesis. LIF is also present in osteoblasts but not in osteoclasts. Observations in a rat endochondral ossification model were confirmed by studies of human cartilage biopsies from foetuses with osteogenesis imperfecta. LIF was never detected in adult articular chondrocytes and bone-marrow mesenchymal cells. These results and other data in the literature suggest that LIF is involved in the delicate balance between the rate of formation of calcified cartilage and its vascularization for bone development.

Age- and genotype-dependence of bone material properties in the osteogenesis imperfecta murine model (oim)

Cortical mineralization of long bones was studied in collagen α2(I)-deficient mice (oim) used as a model for human osteogenesis imperfecta. Aspects of the age development of the mice were characterized by combining nanometer- to micrometer-scale structural analysis with microhardness measurements. Bone structure was determined from homozygous (oim/oim) and heterozygous (oim/+) mice and their normal (+/+) littermates as a function of animal age by small-angle X-ray scattering (SAXS) and quantitative backscattered electron imaging (qBEI) measurements. SAXS studies found anomalies in the size and arrangement of bone mineral crystals in both homozygous and heterozygous mice aged 1–14 months. Generally, the crystals were smaller in thickness and less well aligned in these mice compared with control animals. An increase in the mean crystal thickness of the bone was found within all three genotypes up to an age of 3 months. Vicker’s hardness measurements were significantly enhanced for oim bone (homozygotes and heterozygotes) compared with controls. The microhardness values were correlated directly with increased mineral content of homozygous and heterozygous compared with control bone, as determined by qBEI analysis. There was also a significant increase of mineral content with age. Two possibilities for collagen-mineral association are discussed for explaining the increased hardness and mineral content of oim/oim bone, together with its decreased toughness and thinner mineral crystals. As a consequence of the present measurements, one model for oim bone could incorporate small and densely packed mineral crystals. A second model for possible collagen-mineral association in oim material would consist of two families of mineral crystals, one being smaller and the other being much larger than the crystals found in normal mouse long bones.