Characterisation of ovine bone marrow-derived stromal cells (oBMSC) and evaluation of chondrogenically induced micro-pellets for cartilage tissue repair in vivo.

Bone marrow stromal cells (BMSC) show promise in cartilage repair, and sheep are the most common large animal pre-clinical model. OBJECTIVE: The objective of this study was to characterise ovine BMSC (oBMSC) in vitro, and to evaluate the capacity of chondrogenic micro-pellets manufactured from oBMSC or ovine articular chondrocytes (oACh) to repair osteochondral defects in sheep. DESIGN: oBMSC were characterised for surface marker expression using flow cytometry and evaluated for tri-lineage differentiation capacity. oBMSC micro-pellets were manufactured in a microwell platform, and chondrogenesis was compared at 2%, 5%, and 20% O2. The capacity of cartilage micro-pellets manufactured from oBMSC or oACh to repair osteochondral defects in adult sheep was evaluated in an 8-week pilot study. RESULTS: Expanded oBMSC were positive for CD44 and CD146 and negative for CD45. The common adipogenic induction ingredient, 3-Isobutyl-1-methylxanthine (IBMX), was toxic to oBMSC, but adipogenesis could be restored by excluding IBMX from the medium. BMSC chondrogenesis was optimal in a 2% O2 atmosphere. Micro-pellets formed from oBMSC or oACh appeared morphologically similar, but hypertrophic genes were elevated in oBMSC micro-pellets. While oACh micro-pellets formed cartilage-like repair tissue in sheep, oBMSC micro-pellets did not. CONCLUSION: The sensitivity of oBMSC, compared to human BMSC, to IBMX in standard adipogenic assays highlights species-associated differences. Micro-pellets manufactured from oACh were more effective than micro-pellets manufactured from oBMSC in the repair of osteochondral defects in sheep. While oBMSC can be driven to form cartilage-like tissue in vitro, the effective use of these cells in cartilage repair will depend on the successful mitigation of hypertrophy and tissue integration.

Mutations in NOTCH2 in patients with Hajdu-Cheney syndrome.

UNLABELLED: The Hajdu-Cheney syndrome is a very rare disease that affects several organ system, leading to severe osteoporosis and other abnormalities. We describe clinical and genetic findings of nine patients with this disease. INTRODUCTION: The Hajdu-Cheney syndrome (HCS) is a rare autosomal dominant disorder characterized by severe osteoporosis, acroosteolysis of the distal phalanges, renal cysts, and other abnormalities. Recently, heterozygous mutations in NOTCH2 were identified as the cause of HCS. METHODS: Nine patients with typical presentations of HCS took part in this study: five affected patients from two small families and four sporadic cases. Peripheral blood DNA was obtained and exome sequencing performed in one affected individual per family and in all four sporadic cases. Sanger sequencing confirmed mutations in all patients. RESULTS: One of the identified mutations was introduced in a plasmid encoding NOTCH2. Wild-type and mutant NOTCH2 were transiently expressed in HEK293 cells to assess intracellular localization after ligand activation. Deleterious heterozygous mutations in the last NOTCH2 exon were identified in all patients; five of the six mutations were novel. CONCLUSION: Consistent with previous reports, all mutations are predicted to result in a loss of the proline/glutamic acid/serine/threonine sequence, which harbors signals for degradation, therefore suggesting activating mutations. One of the six mutations furthermore predicted disruption of the second nuclear localization signal of NOTCH2, but the mutant revealed normal nuclear localization after transfection, which is consistent with the proposed gain-of-function mechanism as the cause of this autosomal dominant disease. Our findings confirm that heterozygous NOTCH2 mutations are the cause of HCS and expand the mutational spectrum of this disorder.

Mutant DLX 3 disrupts odontoblast polarization and dentin formation.

Tricho-dento-osseous (TDO) syndrome is an autosomal dominant disorder characterized by abnormalities in the thickness and density of bones and teeth. A 4-bp deletion mutation in the Distal-Less 3 (DLX3) gene is etiologic for most cases of TDO. To investigate the in vivo role of mutant DLX3 (MT-DLX3) on dentin development, we generated transgenic (TG) mice expressing MT-DLX3 driven by a mouse 2.3 Col1A1 promoter. Dentin defects were radiographically evident in all teeth and the size of the nonmineralized pulp was enlarged in TG mice, consistent with clinical characteristics in patients with TDO. High-resolution radiography, microcomputed tomography, and SEM revealed a reduced zone of mineralized dentin with anomalies in the number and organization of dentinal tubules in MT-DLX3 TG mice. Histological and immunohistochemical studies demonstrated that the decreased dentin was accompanied by altered odontoblast cytology that included disruption of odontoblast polarization and reduced numbers of odontoblasts. TUNEL assays indicated enhanced odontoblast apoptosis. Expression levels of the apoptotic marker caspase-3 were increased in odontoblasts in TG mice as well as in odontoblastic-like MDPC-23 cells transfected with MT-DLX3 cDNA. Expression of Runx2, Wnt 10A, and TBC1D19 colocalized with DLX3 expression in odontoblasts, and MT-DLX3 significantly reduced expression of all three genes. TBC1D19 functions in cell polarity and decreased TBC1D19 expression may contribute to the observed disruption of odontoblast polarity and apoptosis. These data indicate that MT-DLX3 acts to disrupt odontoblast cytodifferentiation leading to odontoblast apoptosis, and aberrations of dentin tubule formation and dentin matrix production, resulting in decreased dentin and taurodontism. In summary, this TG model demonstrates that MT-DLX3 has differential effects on matrix production and mineralization in dentin and bone and provides a novel tool for the investigation of odontoblast biology.

Development of craniofacial structures in transgenic mice with constitutively active PTH/PTHrP receptor.

Parathyroid hormone (PTH) and parathyroid hormone-related peptide (PTHrP) regulate calcium homeostasis, and PTHrP further regulates growth and development. A transgenic mouse carrying the constitutively active PTH/PTHrP receptor (HKrk-H223R) under the control of the mouse bone and odontoblast-specific alpha1(I) collagen promoter (Col1-caPPR) has been developed to demonstrate the complex actions of this mutant receptor in hard tissue formation. We have further characterized Col1-caPPR mice abnormalities in the craniofacial region as a function of development. Col1-caPPR mice exhibited a delay in embryonic bone formation, followed by expansion of a number of craniofacial bones including the maxilla and mandible, delay in tooth eruption and teratosis, and a disrupted temporomandibular joint (TMJ). These findings suggest that the Col1-caPPR mouse is a useful model for characterization of the downstream effects of the constitutively active receptor during development and growth, and as a model for development of treatments of human diseases with similar characteristics.

The efficacy of mesenchymal stem cells to regenerate and repair dental structures.

OBJECTIVES: Identification, characterization, and potential application of mesenchymal stem cells (MSC) derived from human dental tissues. METHODS: Dental pulp and periodontal ligament were obtained from normal human impacted third molars. The tissues were digested in collagenase/dispase to generate single cell suspensions. Cells were cultured in alpha-MEM supplemented with 20% fetal bovine serum, 2 mM l-glutamine, 100 microM l-ascorbate-2-phosphate. Magnetic and fluorescence activated cell sorting were employed to characterize the phenotype of freshly isolated and ex vivo expanded cell populations. The developmental potential of cultured cells was assessed following co-transplantation with hydroxyapetite/tricalcium phosphate (HA/TCP) particles into immunocompromised mice for 8 weeks. RESULTS: MSC were identified in adult human dental pulp (dental pulp stem cells, DPSC), human primary teeth (stem cells from human exfoliated deciduous teeth, SHED), and periodontal ligament (periodontal ligament stem cells, PDLSC) by their capacity to generate clongenic cell clusters in culture. Ex vivo expanded DPSC, SHED, and PDLSC populations expressed a heterogeneous assortment of makers associated with MSC, dentin, bone, smooth muscle, neural tissue, and endothelium. PDLSC were also found to express the tendon specific marker, Scleraxis. Xenogeneic transplants containing HA/TCP with either DPSC or SHED generated donor-derived dentin-pulp-like tissues with distinct odontoblast layers lining the mineralized dentin-matrix. In parallel studies, PDLSC generated cementum-like structures associated with PDL-like connective tissue when transplanted with HA/TCP into immunocompromised mice. CONCLUSION: Collectively, these data revealed the presence of distinct MSC populations associated with dental structures with the potential of stem cells to regenerate living human dental tissues in vivo.

Identification of differentially expressed genes between osteoarthritic and normal trabecular bone from the intertrochanteric region of the proximal femur using cDNA microarray analysis.

Osteoarthritis (OA) is a common age-related joint disease resulting in progressive degenerative damage to articular cartilage. The etiology of primary OA has not yet been determined. However, there is evidence supporting the hypothesis that primary OA is a disease affecting bone remodeling in addition to articular cartilage. In this study, we have used cDNA microarray analysis to compare gene expression in bone between normal (CTL) and OA individuals. Trabecular bone was sampled from the intertrochanteric region of the proximal femur, a site distal to the diseased hip joint. Total RNA was extracted from three pairs of age- and sex-matched CTL and OA bone samples, reverse-transcribed and radioactively labeled to generate cDNA probes, before hybridization with the Research Genetics GF211 human gene microarray filter. The CTL and OA samples were found to have similar levels of gene expression for more than 4000 known human genes. However, forty-one genes were identified that were differentially expressed, twofold or more, between all three CTL-OA sample pairs. Using semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) analysis, three genes, fms-like tyrosine kinase 1 (FLT1), plexin B1 (PLXNB1), and small inducible cytokine A2 (SCYA2), were confirmed to be consistently expressed at lower levels in OA, in a majority of twenty age- and sex-matched CTL-OA bone sample pairs tested. FLT1, PLXNB1, and SCYA2 have known or potential roles in angiogenesis and bone remodeling. Down-regulation of these genes is consistent with a role for bone in the pathogenesis of OA.

Analyses of variable panoramic radiographic characteristics of maxillo-mandibular fibrous dysplasia in McCune-Albright syndrome.

OBJECTIVE: Fibrous dysplasia (FD) is a rare skeletal disease caused by activating GNAS1 gene mutations often found in association with the McCune-Albright syndrome (MAS). Multiple bones may be affected in FD, including maxilla and mandible. Patients with MAS have different endocrinopathies that can further influence bone metabolism. The purposes of this cross-sectional study are to characterize FD panoramic radiographic patterns, and to evaluate the effects of age, endocrinopathies and renal phosphate wasting on radiographic characteristics of maxillo-mandibular FD in MAS. SUBJECTS AND METHODS: Fifty-one consecutive MAS patients were screened and panoramic radiographs of 43 patients with craniofacial FD were evaluated and analyzed for FD involvement. Clinical chemistries were evaluated for associations between radiographic patterns and age, endocrinopathies or renal phosphate wasting using Fisher's Exact Test. RESULTS: Four types of radiographic changes were observed: ground glass (granular/condensed trabeculae), radiolucent (lytic), mixed radiolucent/radio-opaque (mixed density) or radio-opaque (sclerotic). Masking or displacement of the maxillary sinus (range: 77.8-86.4%) and mandibular canal (range: 55.6-75.0%) were prevalent in FD sites. Sixty-three percent of the MAS patients had multiple dysregulated endocrine/metabolic functions, the most common were hyperthyroidism, precocious puberty and renal phosphate wasting. There were no statistically significant associations between radiographic patterns and age, endocrinopathies or renal phosphate wasting. CONCLUSIONS: Maxillo-mandibular FD images in panoramic radiographs fall within a spectrum of four different patterns. Patients with facial asymmetry and any of these radiographic patterns should be promptly referred for further radiographic tests and endocrine evaluation if MAS is suspected.

Comparison of human dental pulp and bone marrow stromal stem cells by cDNA microarray analysis.

We compared the gene expression profiles of human dental pulp stem cells (DPSCs) and bone marrow stromal stem cells (BMSSCs) as representative populations of odontoprogenitor and osteoprogenitor cells, respectively. Total RNA from primary cultures was reverse-transcribed to generate cDNA probes and then hybridized with the Research Genetics human gene microarray filter GF211. The microarrays were analyzed using the PATHWAYS software package. Human DPSCs and BMSSCs were found to have a similar level of gene expression for more than 4000 known human genes. A few differentially expressed genes, including collagen type XVIII alpha1, insulin-like growth factor-2 (IGF-2), discordin domain tyrosine kinase 2, NAD(P)H menadione oxidoreductase, homolog 2 of Drosophila large disk, and cyclin-dependent kinase 6 were highly expressed in DPSCs, whereas insulin-like growth factor binding protein-7 (IGFBP-7), and collagen type I alpha2 were more highly expressed in BMSSCs. Furthermore, we confirmed the differential expression of these genes by semiquantitative polymerase chain reaction (PCR) and northern blot hybridization. The protein expression patterns for both IGF-2 and IGFBP-7 correlated with the differential mRNA levels seen between DPSCs and BMSSCs. This report describes the gene expression patterns of two distinct precursor populations associated with mineralized tissue, and provides a basis for further characterization of the functional roles for many of these genes in the development of dentin and bone.

Stem cells in tissue engineering.

The concept of producing 'spare parts' of the body for replacement of damaged or lost organs lies at the core of the varied biotechnological practices referred to generally as tissue engineering. Use of postnatal stem cells has the potential to significantly alter the perspective of tissue engineering. Successful long-term restoration of continuously self-renewing tissues such as skin, for example, depends on the use of extensively self-renewing stem cells. The identification and isolation of stem cells from a number of tissues provides appropriate targets for prospective gene therapies.

Surface protein characterization of human adipose tissue-derived stromal cells.

Human bone marrow stromal cells are a multipotent population of cells capable of differentiating into a number of mesodermal lineages as well as supporting hematopoeisis. Their distinct protein and gene expression phenotype is well characterized in the literature. Human adipose tissue presents an alternative source of multipotent stromal cells. In this study, we have defined the phenotype of the human adipose tissue-derived stromal cells in both the differentiated and undifferentiated states. Flow cytometry and immunohistochemistry show that human adipose tissue-derived stromal cells have a protein expression phenotype that is similar to that of human bone marrow stromal cells. Expressed proteins include CD9, CD10, CD13, CD29, CD34, CD44, CD 49(d), CD 49(e), CD54, CD55, CD59, CD105, CD106, CD146, and CD166. Expression of some of these proteins was further confirmed by PCR and immunoblot detection. Unlike human bone marrow-derived stromal cells, we did not detect the STRO-1 antigen on human adipose tissue-derived stromal cells. Cells cultured under adipogenic conditions uniquely expressed C/EBPalpha and PPARdelta, two transcriptional regulators of adipogenesis. Cells cultured under osteogenic conditions were more likely to be in the proliferative phases of the cell cycle based on flow cytometric analysis of PCNA and Ki67. The similarities between the phenotypes of human adipose tissue-derived and human bone marrow-derived stromal cells could have broad implications for human tissue engineering.

Gnathodiaphyseal dysplasia: a syndrome of fibro-osseous lesions of jawbones, bone fragility, and long bone bowing.

We report an unusual generalized skeletal syndrome characterized by fibro-osseous lesions of the jawbones with a prominent psammomatoid body component, bone fragility, and bowing/sclerosis of tubular bones. The case fits with the emerging profile of a distinct syndrome with similarities to previously reported cases, some with an autosomal dominant inheritance and others sporadic. We suggest that the syndrome be named gnathodiaphyseal dysplasia. The patient had been diagnosed previously with polyostotic fibrous dysplasia (PFD) elsewhere, but further clinical evaluation, histopathological study, and mutation analysis excluded this diagnosis. In addition to providing a novel observation of an as yet poorly characterized syndrome, the case illustrates the need for stringent diagnostic criteria for FD. The jaw lesions showed fibro-osseous features with the histopathological characteristics of cemento-ossifying fibroma, psammomatoid variant. This case emphasizes that the boundaries between genuine GNAS1 mutation-positive FD and other fibro-osseous lesions occurring in the jawbones should be kept sharply defined, contrary to a prevailing tendency in the literature. A detailed pathological study revealed previously unreported features of cemento-ossifying fibroma, including the participation of myofibroblasts and the occurrence of psammomatoid bodies and aberrant mineralization, within the walls of blood vessels. Transplantation of stromal cells grown from the lesion into immunocompromised mice resulted in a close mimicry of the native lesion, including the sporadic formation of psammomatoid bodies, suggesting an intrinsic abnormality of bone-forming cells.

Renal phosphate wasting in fibrous dysplasia of bone is part of a generalized renal tubular dysfunction similar to that seen in tumor-induced osteomalacia.

Fibrous dysplasia (FD) of bone is characterized by focal replacement of normal bone and marrow with abnormal bone and fibrous tissue. It arises from postzygotic activating mutations of the GNAS1 gene. Hypophosphatemia due to renal phosphate wasting has been reported in association with FD as a part of the McCune-Albright Syndrome (MAS), which is characterized by FD, skin hyperpigmentation, and precocious puberty. To date, the prevalence and mechanism of phosphate wasting has not been well studied. We evaluated 42 patients with FD/MAS. Serum and urine samples were tested for indices of mineral metabolism, amino acid handling, and markers of bone metabolism. Twenty (48%) patients had some degree of renal phosphate wasting. Nephrogenous cyclic adenosine monophosphate (cAMP) was normal in FD patients, suggesting that the underlying cause of phosphate wasting is not the presence of activating GNAS1 mutations in the kidney. In addition, there was evidence of a more generalized renal tubulopathy as represented by the presence of abnormal vitamin D metabolism, proteinuria in 36 (86%) patients, and aminoaciduria in 39 (94%) patients. Renal phosphate wasting significantly correlated with the degree of bone involvement, as assessed by serum and urine markers of bone metabolism, suggesting that a circulating factor produced by FD bone and impacting on the kidney may be the mechanism. These data show that phosphaturia as part of a generalized renal tubulopathy represents the most common extraskeletal manifestation of FD and that the observed tubulopathy is similar to that seen in tumor-induced osteomalacia (TIO).

Bone marrow stromal stem cells: nature, biology, and potential applications.

Bone marrow stromal cells are progenitors of skeletal tissue components such as bone, cartilage, the hematopoiesis-supporting stroma, and adipocytes. In addition, they may be experimentally induced to undergo unorthodox differentiation, possibly forming neural and myogenic cells. As such, they represent an important paradigm of post-natal nonhematopoietic stem cells, and an easy source for potential therapeutic use. Along with an overview of the basics of their biology, we discuss here their potential nature as components of the vascular wall, and the prospects for their use in local and systemic transplantation and gene therapy.

Circulating skeletal stem cells.

We report the isolation of adherent, clonogenic, fibroblast-like cells with osteogenic and adipogenic potential from the blood of four mammalian species. These cells phenotypically resemble but are distinguishable from skeletal stem cells found in bone marrow (stromal stem cells, "mesenchymal stem cells"). The osteogenic potential of the blood-borne cells was proven by an in vivo transplantation assay in which either polyclonal or single colony-derived strains were transplanted into the subcutis of immunocompromised mice, and the donor origin of the fully differentiated bone cells was proven using species-specific probes. This is the first definitive proof of the existence of circulating skeletal stem cells in mammals.

Pedicled bone flap formation using transplanted bone marrow stromal cells.

HYPOTHESIS: Transplanted osteoprogenitor cells derived from cultured bone marrow stromal cells (BMSCs) can be used to fabricate pedicled bone flaps. DESIGN: Prospective, randomized experimental trials. SETTING: Basic science research laboratory. MATERIALS: Immunodeficient female NIH-Bg-Nu-Xid mice, aged 3 months. INTERVENTION: The BMSCs were harvested from the long bones of C57Bl/6 transgenic mice carrying the type Ialpha1 collagen-chloramphenicol acetyl transferase reporter gene construct; their numbers were expanded in tissue culture. Treated mice received BMSC transplantations around the common carotid artery and internal jugular vein, the aorta and its venae comitantes, or the saphenous artery and vein; control mice received a sham transplant in comparable recipient sites. MAIN OUTCOME MEASURES: Mice underwent harvesting from 4 weeks to 2 years after transplantation. Transplants were evaluated via histological, immunohistochemical, and angiographic analyses. RESULTS: Compared with the controls, which formed no bone, 32 of 37 BMSC-containing transplants formed a vascularized bone island that was perfused specifically and solely by its common carotid artery vascular source. Mature transplants consisted of well-developed lamellar, corticocancellous bone whose osteocytes were derived from the grafted BMSCs; hematopoietic tissue derived from the recipient mouse. Transplants formed as early as 4 weeks and remained stable in size as late as 108 weeks. CONCLUSIONS: Bone marrow stromal cells can be used to create vascularized bone flaps in mice; these bone constructs are vascularized by their pedicle and therefore can potentially be transferred to a recipient site using microsurgical techniques. These findings provide proof of principle of an additional clinical application of BMSC transplantation techniques.

Integrin-mediated interactions between human bone marrow stromal precursor cells and the extracellular matrix.

To date, the precise interactions between bone marrow stromal cells and the extracellular matrix that govern stromal cell development remain unclear. The integrin super-family of cell-surface adhesion molecules represents a major pathway used by virtually all cell types to interact with different extracellular matrix components. In this study, purified populations of stromal precursor cells were isolated from the STRO-1-positive fraction of normal human marrow, by fluoresence-activated cell sorting, and then assayed for their ability to initiate clonogenic growth in the presence of various integrin ligands. Bone marrow-derived stromal progenitors displayed differential growth to fibronectin, vitronectin, and laminin, over collagen types I and III, but showed a similar affinity for collagen type IV. The integrin heterodimers alpha1beta1, alpha2beta1, alpha5beta1, alpha6beta1, alpha(v)beta3, and alpha(v)beta5 were found to coexpress with the STRO-1 antigen on the cell surface of CFU-F, using dual-color analysis. Furthermore, only a proportion of stromal precursors expressed the integrin alpha4beta1, while no measurable levels of the integrin alpha3beta1 could be detected. Subsequent adhesion studies using functional blocking antibodies to different integrin alpha/beta heterodimers showed that stromal cell growth on collagen, laminin, and fibronectin was mediated by multiple beta1 integrins. In contrast, cloning efficiency in the presence of vitronectin was mediated in part by alpha(v)beta3. When human marrow stromal cells were cultured under osteoinductive conditions, their ability to form a mineralized matrix in vitro was significantly diminished in the presence of a functional blocking monoclonal antibody to the beta1 integrin subunit. The results of this study indicate that beta1 integrins appear to be the predominant adhesion receptor subfamily utilized by stromal precursor cells to adhere and proliferate utilizing matrix glycoproteins commonly found in the bone marrow microenvironment and bone surfaces. Furthermore, these data suggest a possible role for the beta1 integrin subfamily during the development of stromal precursor cells into functional osteoblast-like cells.

In vivo bone formation by human bone marrow stromal cells: effect of carrier particle size and shape.

Successful closure of bone defects in patients remains an active area of basic and clinical research. A novel and promising approach is the transplantation of human bone marrow stromal cells (BMSCs), which have been shown to possess a significant osteogenic potential. The extent and quality of bone formation by transplanted human BMSCs strongly depends on the carrier matrix with which cells are transplanted; to date, hydroxyapatite/tricalcium phosphate (HA/TCP) supports far more osteogenesis than any other matrix tested. In order to further improve the technique of BMSC transplantation, we studied whether commercially available HA/TCP particles, clinically approved as an osteoconductive material and commercially available as particles measuring 0.5-1.0 mm diameter, is an optimum matrix for promoting bone development by BMSCs. HA/TCP and HA particles of varying size were sieved into a variety of size ranges, from <0.044 mm to 1.0-2.0 mm. Transplants were formed by mixing 40 mg aliquots of particles with cultured passaged human BMSCs. They were placed in subcutaneous pockets in immunocompromised Bg-Nu-XID mice and harvested 4 or 10 weeks later. The transplants were examined histologically; the presence of bone within each transplant was evaluated using histomorphometry or blindly scored on a semiquantitative scale. Transplant morphology and the amount of new bone varied in a consistent fashion based on particle size and shape. Transplants incorporating HA/TCP particles of 0.1-0.25 mm size demonstrated the greatest bone formation at both 4 and 10 weeks; larger or smaller particles were associated with less extensive bone formation, while a size of 0.044 mm represented a threshold below which no bone formation could be observed. Flat-sided HA particles measuring 0.1-0.25 mm formed no bone. The differences in bone formation were not attributable to the differences in cell attachment among the groups. Instead, the size and spatial and structural organization of the particles within BMSC transplants appear to determine the extent of bone formation. These findings provide necessary information for the successful clinical application of BMSC transplantation techniques.