In recent years, translational research with various animal models has been helpful to answer basic questions about the effect of antenatal inflammation on maturation and development of the fetal lung and immune system. The fetal lung and immune systems are very plastic and their development can be conditioned and influenced by both endogenous and/or exogenous factors. Antenatal inflammation can induce pulmonary inflammation, leading to lung injury and remodeling in the fetal lung. Exposure to antenatal inflammation can induce interleukin-1α production, which enhances surfactant <em>protein</em> and lipid synthesis thereby promoting lung maturation. Interleukin-1α is therefore a candidate for the link between lung inflammation and lung maturation, preventing respiratory distress syndrome in preterm infants. Antenatal inflammation can, however, cause structural changes in the fetal lung and affect the expression of growth factors, such as transforming growth factor-beta, connective tissue growth factor, fibroblast growth factor-<em>10</em>, or <em>bone</em> <em>morphogenetic</em> <em>protein</em>-4, which are essential for branching morphogenesis. These alterations cause alveolar and microvascular simplification resembling the histology of bronchopulmonary dysplasia. Antenatal inflammation may also affect neonatal outcome by modulating the responsiveness of the immune system. Lipopolysaccharide-tolerance (endotoxin hyporesponsiveness/immunoparalysis), induced by exposure to inflammation in utero, may prevent fetal lung damage, but increases susceptibility to postnatal infections. Moreover, prenatal exposure to inflammation appears to be a predisposition for the development of adverse neonatal outcomes, like bronchopulmonary dysplasia, if the preterm infant is exposed to a second postnatal hit, such as mechanical ventilation oxygen exposure, infections, or steroids.

Most recombinant human <em>bone</em> <em>morphogenetic</em> <em>protein</em> (rhBMP) is currently obtained from Chinese hamster ovary (CHO) cells. If rhBMP with more activity could be derived from Escherichia coli (E. coli), a large quantity of rhBMP could be produced at low cost. The <em>bone</em>-inducing ability of an E. coli -derived rhBMP-2 (ErhBMP-2) variant with an N-terminal sequence was examined and compared with CHO cell-derived rhBMP-2 (CrhBMP-2). Two, <em>10</em>, or 50 microgram of ErhBMP-2 or CrhBMP-2 was mixed with 3mg of atelopeptide type I collagen as the carrier, and specimens were implanted into the calf muscle pouches of Wistar rats (n= 5 in each group). Three weeks later, new <em>bone</em> had formed in all the ErhBMP-2-implanted and CrhBMP-2-implanted muscles. Radiographic and histological examinations showed that the <em>bone</em> induced by ErhBMP-2 had a large hollow <em>bone</em> matrix with more fatty marrow than the <em>bone</em> induced by CrhBMP-2. Quantitative analysis indicated that the activity of ErhBMP-2 was similar to that of CrhBMP-2, so ErhBMP-2 may be useful for inducing <em>bone</em> formation.

BACKGROUND

A cryopreservation protocol for human amnion-derived mesenchymal stem cells (HAMs) is required because these cells cannot survive for long periods in culture. The aim of this study was to determine whether vitrification is a useful freezing method for storage of HAMs.

METHODS

HAMs were cryopreserved using vitrification method. The morphology and viability of thawed HAMs was evaluated by Trypan Blue staining. The expression of several embryonic stem cell (ESC) markers was evaluated using flow cytometry, RT-PCR and immunocytochemistry. Von Kossa, Oil Red O and Alcian Blue staining were used to asses the differentiation potential of thawed HAMs.

RESULTS

The post-thawing viability of HAMs was 84.3 +/- 3.2% (Mean +/- SD, n = <em>10</em>). The thawed HAMs showed morphological characteristics indistinguishable from the non-vitrified fresh HAMs. The expression of surface antigens (strong positive for CD44, CD49d, CD59, CD90, CD<em>10</em>5 and HLA-ABC; weak positive for HLA-G; negative for CD31, CD34, CD45, CD<em>10</em>6, CD117 and HLA-DR) and the expression of ESC markers [CK18, fibroblast growth factor-5, GATA-4, neural cell adhesion molecule, Nestin, Oct-4, stem cell factor, HLA-ABC, Vimentin, <em>bone</em> <em>morphogenetic</em> <em>protein</em> (BMP) 4, hepatocyte nuclear factor 4 alpha (HNF-4 alpha), Pax-6, alpha-feto<em>protein</em>, Brachyury, BMP-2, TRA-1-60, stage-specific embryonic antigen (SSEA-3, SSEA-4)] were maintained in the vitrified-thawed HAMs. The thawed HAMs retained ability to differentiate into osteoblasts, adipocytes and chondrocytes under appropriate culture conditions.

CONCLUSIONS

Our results suggest that vitrification is a reliable and effective method for cryopreservation of HAMs.

Since their discovery, <em>bone</em> <em>morphogenetic</em> <em>proteins</em> have held the promise for use in various orthopaedic diseases. One of the largest areas of likely application is the area of fracture repair. Although millions of fractures occur annually and the majority heal satisfactorily, 5% to <em>10</em>% go on to delayed union or nonunion. <em>Bone</em> <em>morphogenetic</em> <em>proteins</em> may be able to improve bony healing in these conditions and perhaps enhance the healing of fractures that otherwise heal satisfactorily. This study examines the preclinical data to support the concept of enhancing bony healing and discusses the preliminary data from clinical trials using <em>bone</em> <em>morphogenetic</em> <em>proteins</em> to augment bony healing. Although the potential clinical uses of <em>bone</em> <em>morphogenetic</em> <em>proteins</em> in fracture healing remain significant, this potential has yet to be realized.

The morphological stages of tooth development--bud, cap, bell, and terminal differentiation--have been known for decades. The past <em>10</em> years have seen the elucidation of many of the molecular events driving these <em>morphogenetic</em> stages. Signaling via the fibroblast growth factor (FGF), <em>bone</em> <em>morphogenetic</em> <em>protein</em> (BMP), hedgehog, and wingless <em>protein</em> families and their downstream transcription factors have been identified as key players in the epithelial-mesenchymal signaling loops driving tooth development. Currently the most complete description of the mechanisms in tooth development extends only through the cap stage. The body of work concerning the mechanisms directing the bell and cytodifferentiation stages is growing. This has mainly, but not exclusively, focused on the expression and effects of FGFs and BMPs in these latter stages, and is reviewed here. Additionally, recent results suggest that phenotypic <em>proteins</em> of both ameloblasts and odontoblasts, such as amelogenin and dentin matrix <em>protein</em> 2 may act as the final instructive signals in cytodifferentiation.

In developing new generations of coatings for medical devices and tissue engineering scaffolds, there is a need for thin coatings that provide controlled sequential release of multiple therapeutics while providing a tunable approach to time dependence and the potential for sequential or staged release. Herein, we demonstrate the ability to develop a self-assembled, polymer-based conformal coating, built by using a water-based layer-by-layer (LbL) approach, as a dual-purpose biomimetic implant surface that provides staggered and/or sustained release of an antibiotic followed by active growth factor for orthopedic implant applications. This multilayered coating consists of two parts: a base osteoinductive component containing <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 (rhBMP-2) beneath an antibacterial component containing gentamicin (GS). For the fabrication of truly stratified composite films with the customized release behavior, we present a new strategy-implementation of laponite clay barriers-that allows for a physical separation of the two components by controlling interlayer diffusion. The clay barriers in a single-component GS system effectively block diffusion-based release, leading to approximately 50% reduction in bolus doses and <em>10</em>-fold increase in the release timescale. In a dual-therapeutic composite coating, the top GS component itself was found to be an effective physical barrier for the underlying rhBMP-2, leading to an order of magnitude increase in the release timescale compared to the single-component rhBMP-2 system. The introduction of a laponite interlayer barrier further enhanced the temporal separation between release of the two drugs, resulting in a more physiologically appropriate dosing of rhBMP-2. Both therapeutics released from the composite coating retained their efficacy over their established release timeframes. This new platform for multi-drug localized delivery can be easily fabricated, tuned, and translated to a variety of implant applications where control over spatial and temporal release profiles of multiple drugs is desired.

Activins and inhibins, members of the transforming growth factor-beta family are able to stimulate and inhibit, respectively, FSH synthesis and release. Other members of this superfamily, the <em>bone</em> <em>morphogenetic</em> <em>proteins</em> (BMPs), may also affect FSH synthesis in the mouse. The aim of this work was to determine whether BMPs are expressed in the ovine pituitary and whether they play a role in the regulation of FSH release. The mRNAs encoding BMP-2, BMP-4, BMP-7 and the oocyte-derived growth factor, growth differentiation factor (GDF)-9 were detected in the pituitaries of cyclic ewes by reverse-transcriptase PCR, as well as the mRNAs encoding the BMP type I receptors, BMPR-IA (activin-receptor-like kinase (ALK)-3) and BMPR-IB (ALK-6), and type II receptors (BMPR-II). Immunolabeling of pituitary sections revealed the presence of BMPR-IA (ALK-3) and BMPR-II in gonadotrope cells. To investigate the potential effects of BMPs on FSH secretion, ewe pituitary cell cultures were treated with BMP-4 (<em>10</em>(-11) M to <em>10</em>(-9) M) for 48 h. Interestingly, FSH release was decreased in a dose-dependent manner. At <em>10</em>(-9) M BMP-4 both FSH concentration and FSHbeta mRNA expression were reduced by 40% of control values. In contrast, there was no inhibitory effect on either LH or LHbeta mRNA expression. A similar result was found with BMP-6. BMP-4 triggered the phosphorylation of Smad1, suggesting that the effect of BMP-4 on FSH secretion is due to the activation of the BMPs signaling pathway. Furthermore, BMP-4 blocked the stimulatory effect of activin on both FSH release and FSHbeta mRNA and amplified the suppression of FSH release and FSHbeta mRNA levels induced by 17beta-estradiol. These results indicate that a functional BMP system operates within the sheep pituitary, at least in vitro, to decrease FSH release and to modulate the effect of activin.

A cDNA clone coding for a sea urchin embryonic <em>protein</em> was isolated from a prehatching blastula lambda gt11 library. The predicted translation product is a secreted 64 x <em>10</em>(3) Mr enzyme designated as BP<em>10</em>. The <em>protein</em> contains several domains: a signal peptide, a putative propeptide, a catalytic domain with an active center typical of a Zn(2+)-metalloprotease, an EGF-like domain and two internal repeats similar to repeated domains found in the C1s and C1r serine proteases of the complement cascade. The BP<em>10</em> protease is constructed with the same domains as the human <em>bone</em> <em>morphogenetic</em> <em>protein</em> BMP-1, a protease described as a factor involved in <em>bone</em> formation, and as the recently characterized product of the tolloid gene which is required for correct dorsal-ventral patterning of the Drosophila embryo. The transcription of the BP<em>10</em> gene is transiently activated around the 16- to 32-cell stage and the accumulation of BP<em>10</em> transcripts is limited to a short period at the blastula stage. By in situ hybridization with digoxygenin-labelled RNA probes, the BP<em>10</em> transcripts were only detected in a limited area of the blastula, showing that the transcription of the BP<em>10</em> gene is also spatially controlled. Antibodies directed against a fusion <em>protein</em> were used to detect the BP<em>10</em> <em>protein</em> in embryonic extracts. The <em>protein</em> is first detected in early blastula stages, its level peaks in late cleavage, declines abruptly before ingression of primary mesenchyme cells and remains constant in late development. The distribution of the BP<em>10</em> <em>protein</em> during its synthesis and secretion was analysed by immunostaining blastula-stage embryos. The intracellular localization of the BP<em>10</em> staining varies with time. The <em>protein</em> is first detected in a perinuclear region, then in an apical and submembranous position just before its secretion into the perivitelline space. The <em>protein</em> is synthesized in a sharply delimited continuous territory spanning about 70% of the blastula. Comparison of the size and orientation of the labelled territory in the late blastula with the fate map of the blastula stage embryo shows that the domain in which the BP<em>10</em> gene is expressed corresponds to the presumptive ectoderm. Developing embryos treated with purified antibodies against the BP<em>10</em> <em>protein</em> and with synthetic peptides derived from the EGF-like domain displayed perturbations in morphogenesis and were radialized to various degrees. These results are consistent with a role for BP<em>10</em> in the differentiation of ectodermal lineages and subsequent patterning of the embryo. On the basis of these results, we speculate that the role of BP<em>10</em> in the sea urchin embryo might be similar to that of tolloid in Drosophila. We discuss the idea that the processes of spatial regulation of gene expression along the animal-vegetal in sea urchin and dorsal-ventral axes in Drosophila might have some similarities and might use common elements.

The present investigation was performed to study the adsorption behavior of growth factors and their release characteristics from biodegradable implants in an in vitro study. We investigated the stability of growth factors administered on various scaffolds. We used porous tricalcium phosphate ceramics (alpha-TCP), a neutralized glass-ceramics (GB9N), a composite (polylactid/-glycolid/GB9N), and solvent dehydrated human <em>bone</em> as carriers. Block shaped scaffolds (sized: 7 x 7 x <em>10</em> mm) were loaded with 5 microg of either <em>bone</em> <em>morphogenetic</em> <em>protein</em> (rxBMP-4), basic fibroblast growth factor (rh-bFGF), or vascular endothelial growth factor (rh-VEGF) solved in 150 microL PBS. The growth factors were labeled with Iodine125 (I-125) for detecting the adsorbed and released amount of growth factors by counting the samples for total I-125 activity. We observed that the adsorption of these growth factors seems to depend on two different parameters: first on the nature of the tested material, and second on the growth factors on their own. The release kinetics of the growth factors from the biodegradable implants can be described as a two phase process-a very rapid release during the first hours by an elution of not adsorbed <em>protein</em>, followed by a specific release, which depends upon the chemical/physical interaction of the material and the growth factor used. Analyzing the eluted <em>proteins</em> on SDS-PAGEs rh-VEGF was degraded into a smaller fragment with a size of around 15 kDa, while rxBMP-4 and rh-bFGF showed a complete degradation into fragments smaller than 3 kDa after more than 3 days. Although this in vitro study suggests that biodegradable implants might be successfully used as carriers for osteogenic growth factors, the different release kinetics as well as the alteration of their molecular structure including loss of biological activity should be considered.

BACKGROUND

The molecular pathogenesis of diabetic glomerulosclerosis remains unknown, but recent studies suggest that podocyte damage may play a role. Bone morphogenetic protein 7 (BMP-7) is physiologically expressed in podocytes and tubular epithelial cells. Our previous studies show that BMP-7 reverses glomerular and tubulointerstitial damage in diabetic rats, but there is little known about possible effects of BMP-7 on podocytes. We postulate that high glucose may injure the podocyte by altering structural proteins such as synaptopodin and podocin. This study investigates the effect of high glucose on mouse podocytes, expression of synaptopodin and podocin under normal and high glucose and the treatment effect of BMP-7 on these molecules. Human diabetic glomeruli are studied in parallel.

METHODS

Conditionally immortalized mouse podocytes were exposed to media containing normal (NG) or high (HG) glucose for 2 weeks. Synaptopodin, podocin and BMP-7 gene transcription and protein were assayed with real-time PCR, Western blot or immunohistochemistry, respectively. Synaptopodin and podocin mRNA and protein was evaluated using podocytes incubated in HG for 1 week, in the presence of low (10 ng/ml) and high (300 ng/ml) dose recombinant BMP-7 (rhBMP-7). Human diabetic glomeruli were excised from renal biopsies by laser capture micro-dissection (LCM) and endogenous BMP7 and synaptopodin and podocin were determined by RT-PCR and/or immunohistochemistry.

RESULTS

Culture of podocytes in HG decreases synaptopodin, podocin and BMP-7 transcription and protein synthesis compared to NG. Treatment with rhBMP-7 restores synaptopodin and podocin mRNA and protein. Decreased BMP-7 and synaptopodin is also observed in human diabetic glomeruli both at the transcription and protein level.

CONCLUSIONS

BMP-7 may confer resistance to hyperglycaemic injury via synaptopodin and podocin suggesting novel BMP7 therapies for diabetic glomerulosclerosis.

OBJECTIVE

Maternal food restriction during pregnancy results in growth-restricted newborns and reduced glomerular number, contributing to programmed offspring hypertension. We investigated whether reduced nephrogenesis may be programmed by dysregulation of factors controlling ureteric bud branching and mesenchyme to epithelial transformation.

METHODS

At <em>10</em> to 20 days' gestation, Sprague Dawley pregnant rats (n = 6/group) received ad libitum food; food-restricted rats were 50% food restricted. At embryonic day 20, messenger ribonucleic acid (mRNA) and <em>protein</em> expression of Wilms' tumor 1 gene product (WT1), paired box transcription factor (Pax)-2, fibroblast growth factor (FGF)-2, glial cell line-derived neurotrophic factor (GDNF), cRET, wingless-type mouse mammary tumor virus integration site (WNT)4, WNT11, <em>bone</em> <em>morphogenetic</em> <em>protein</em> (BMP)-4, BMP7, and FGF7 were determined by real-time polymerase chain reaction and Western blotting.

RESULTS

Maternal food restriction resulted in up-regulated mRNA expression for WT1, FGF2, and BMP7, whereas Pax2, GDNF, FGF7, BMP4, WNT4, and WNT11 mRNAs were down-regulated. Protein expression was concordant for WT1, GDNF, Pax2, FGF7, BMP4, and WNT4.

CONCLUSIONS

Maternal food restriction altered gene expression of fetal renal transcription and growth factors and likely contributes to development of offspring hypertension.

If cell based therapy for spinal cord injury is to become a reality, greater insights into the biology of human derived spinal cord stem cells are a prerequisite. Significant species differences and regional specification of stem cells necessitates determining the effects of growth factors on human spinal cord stem cells. Fetal spinal cords were dissociated and expanded as neurospheres in medium with <em>bone</em> <em>morphogenetic</em> <em>protein</em> 4 (BMP4), leukemia inhibitory factor (LIF) or BMP4 and LIF. First-generation neurospheres comprised a heterogeneous population of neural cell types and after plating emergent cells included neurons, oligodendrocytes and GFAP(+) cells which coexpressed stem cells markers and those of the neuronal lineage and were thus identified as GFAP(+) neural precursor cells (NPC). When plated, neurospheres maintained in BMP4 demonstrated a reduced proportion of emergent oligodendrocytes from 13 to 4%, whereas LIF had no statistically significant effect on cell type distribution. Combining BMP4 and LIF reduced the proportion of oligodendrocytes to 3% and that of neurons from 37 to 16% while increasing the proportion of GFAP(+) NPC from 45 to 79%. After <em>10</em> passages in control media aggregates gave rise to multiple neural phenotypes and only continued passage of neurospheres in the presence of BMP4 and LIF resulted in unipotent aggregates giving rise to only astrocytes. These results provide a means of obtaining pure populations of human spinal-cord derived astrocytes, which could be utilized for further studies of cell replacement strategies or in vitro evaluation of therapeutics.

The understanding of renal fibrosis in chronic kidney disease (CKD) remains as a challenge. More than <em>10</em>% of the population of developed countries suffer from CKD. Proliferation and activation of myofibroblasts and accumulation of extracellular matrix <em>proteins</em> are the main features of kidney fibrosis, a process in which a large number of cytokines are involved. Targeting cytokines responsible for kidney fibrosis development might be an important strategy to face the problem of CKD. The increasing knowledge of the signaling pathway network of the transforming growth factor beta (TGF-β) superfamily members, such as the profibrotic cytokine TGF-β1 or the <em>bone</em> <em>morphogenetic</em> <em>proteins</em> (BMPs), and their involvement in the regulation of kidney fibrosis, has stimulated numerous research teams to look for potential strategies to inhibit profibrotic cytokines or to enhance the anti-fibrotic actions of other cytokines. The consequence of all these studies is a better understanding of all these canonical (Smad-mediated) and non-canonical signaling pathways. In addition, the different receptors involved for signaling of each cytokine, the different combinations of type I-type II receptors, and the presence and function of co-receptors that can influence the biological response have been also described. However, are these studies leading to suitable strategies to block the appearance and progression of kidney fibrosis? In this review, we offer a critical perspective analyzing the achievements using the most important strategies developed up till now: TGF-β antibodies, chemical inhibitors of TGF-β receptors, miRNAs and signaling pathways and BMP agonists with a potential role as therapeutic molecules against kidney fibrosis.

<em>Bone</em> <em>morphogenetic</em> <em>protein</em> (BMP) pathways control an array of developmental and homeostatic events, and must themselves be exquisitely controlled. Here, we identify Caenorhabditis elegans SMA-<em>10</em> as a positive extracellular regulator of BMP-like receptor signaling. SMA-<em>10</em> acts genetically in a BMP-like (Sma/Mab) pathway between the ligand DBL-1 and its receptors SMA-6 and DAF-4. We cloned sma-<em>10</em> and show that it has fifteen leucine-rich repeats and three immunoglobulin-like domains, hallmarks of an LRIG subfamily of transmembrane <em>proteins</em>. SMA-<em>10</em> is required in the hypodermis, where the core Sma/Mab signaling components function. We demonstrate functional conservation of LRIGs by rescuing sma-<em>10</em>(lf) animals with the Drosophila ortholog lambik, showing that SMA-<em>10</em> physically binds the DBL-1 receptors SMA-6 and DAF-4 and enhances signaling in vitro. This interaction is evolutionarily conserved, evidenced by LRIG1 binding to vertebrate receptors. We propose a new role for LRIG family members: the positive regulation of BMP signaling by binding both Type I and Type II receptors.

OBJECTIVE

The aim of this systematic review was to evaluate clinical and safety data for recombinant human bone morphogenetic protein-2 (rhBMP-2) in an absorbable collagen sponge (ACS) carrier when used for alveolar ridge/maxillary sinus augmentation in humans.

METHODS

Clinical studies/case series published 1980 through June 2012 using rhBMP-2/ACS were searched. Studies meeting the following criteria were considered eligible for inclusion: >10 subjects at baseline and maxillary sinus or alveolar ridge augmentation not concomitant with implant placement.

RESULTS

Seven of 69 publications were eligible for review. rhBMP-2/ACS yielded clinically meaningful bone formation for maxillary sinus augmentation that would allow placement of regular dental implants without consistent differences between rhBMP-2 concentrations. Nevertheless, the statistical analysis showed that sinus augmentation following autogenous bone graft was significantly greater (mean bone height: 1.6 mm, 95% CI: 0.5-2.7 mm) than for rhBMP-2/ACS (rhBMP-2 at 1.5 mg/mL). In extraction sockets, rhBMP-2/ACS maintained alveolar ridge height while enhancing alveolar ridge width. Safety reports did not represent concerns for the proposed indications.

CONCLUSIONS

rhBMP-2/ACS appears a promising alternative to autogenous bone grafts for alveolar ridge/maxillary sinus augmentation; dose and carrier optimization may expand its efficacy, use, and clinical application.

Transcriptional coactivator with PDZ-binding motif (TAZ), a beta-catenin-like molecule, drives mesenchymal stem cell (MSC) to differentiate into osteoblast lineage through co-activation of Runx2-dependent gene transcription and repression of peroxisome proliferator-activated receptorgamma (PPARgamma)-dependent gene transcription. Dexamethasone (DEX), a synthetic and widely used glucocorticoid, affects osteogenesis. However, the signaling pathway by which DEX affects osteoblastic differentiation remains obscure. In this study, we found that DEX at the concentration of <em>10</em>(-8)M enhanced calcium deposition, TAZ, <em>bone</em> <em>morphogenetic</em> <em>protein</em> 2 (BMP-2) and alkaline phosphatase (ALP) expression during osteoblastic differentiation. RU486, an antagonist of glucocorticoid receptor, blocked the improvement of TAZ expression while MSCs were treated with <em>10</em>(-8)M DEX. Moreover, higher concentration (<em>10</em>(-7)M) of DEX robustly suppressed TAZ and ALP expression in MSCs. These findings suggest that TAZ is not only involved in the signal pathway of BMP-2-induced osteoblastic differentiation, but also involved in the signaling pathway of DEX-induced osteoblastic differentiation, supporting the notion that TAZ is a convergence point of two signaling pathways, BMP-2 signaling pathway and Wnt-beta-catenin signaling pathway.

The present study addressed adult human mesenchymal stem cell (MSC) differentiation toward the osteoblastic lineage in response to alternating electric current, a biophysical stimulus. For this purpose, MSCs (chosen because of their proven capability for osteodifferentiation in the presence of select <em>bone</em> <em>morphogenetic</em> <em>proteins</em>) were dispersed and cultured within electric-conducting type I collagen hydrogels, in the absence of supplemented exogenous dexamethasone and/or growth factors, and were exposed to either <em>10</em> or 40 μA alternating electric current for 6 h per day. Under these conditions, MSCs expressed both early- (such as Runx-2 and osterix) and late- (specifically, osteopontin and osteocalcin) osteogenic genes as a function of level, and duration of exposure to alternating electric current. Compared to results obtained after 7 days, gene expression of osteopontin and osteocalcin (late-osteogenic genes) increased at day 14. In contrast, expression of these osteogenic markers from MSCs cultured under similar conditions and time periods, but not exposed to alternating electric current, did not increase as a function of time. Most importantly, expression of genes pertinent to the either adipogenic (specifically, Fatty Acid Binding <em>Protein</em>-4) or chondrogenic (specifically, type II collagen) pathways was not detected when MSCs were exposed to the aforementioned alternating electric-current conditions tested in the present study. The present research study was the first to provide evidence that alternating electric current promoted the differentiation of adult human MSCs toward the osteogenic pathway. Such an approach has the yet untapped potential to provide critically needed differentiated cell supplies for cell-based assays and/or therapies for various biomedical applications.

In this study, we investigated the influence of transforming growth factor beta 3 (TGF-beta3), <em>bone</em> <em>morphogenetic</em> <em>protein</em> 6 (BMP-6) and basic fibroblast growth factor (FGF-2) on chondrogenesis in adipose derived stem cells (ASC). Cells were isolated from liposuction material, expanded and subjected to chondrogenic differentiation. Micromass pellets were cultured in chondrogenic medium containing <em>10</em> ng/mL TGF-beta3 which was additionally supplemented with <em>10</em> ng/mL BMP-6, <em>10</em> ng/mL FGF-2 or a combination of both. We quantitatively evaluated the cartilage specific gene expression after 14 days of culture. The end point measurements on day 35 included glycosaminoglycan (GAG) quantification, histological staining for chondrogenic markers, and transmission electron microscopy (TEM). In comparison to cultures induced with TGF-beta3/FGF-2, the presence of TGF-beta3/BMP-6 demonstrated strong induction of collagen type II, collagen type IX and aggrecan mRNA expression. This was corroborated by quantification and histological staining for GAGs and immunohistological staining for collagen II. However, when a combination of BMP-6 and FGF-2 in addition to TGF-beta3 was added, FGF-2 counteracted BMP-6, as indicated by reduced marker gene expression and weak to absent staining for GAGs. In conclusion, this study demonstrates that BMP-6 combined with TGF-beta3 is a potent inducer of chondrogenesis in human ASC. In contrast, FGF-2 does not contribute to differentiation, but rather suppresses the chondrogenic potential of BMP-6.

The aim of the present study was to investigate the possibility of integrating porous hydroxyapatite (HA) ceramics with a capillary vessel network via insertion of a vascular pedicle, and to determine whether this procedure enhances new <em>bone</em> formation in tissue engineering of <em>bone</em>. First, synthetic interconnected porous HA (IP-CHA) was implanted subcutaneously into rat groin with or without insertion of superficial inferior epigastric vessels. At 6 weeks, IP-CHA with vascular insertion contained thick fibrous connective tissue with a number of large blood vessels that seemed to derive from the inserted vascular bundle. Next, IP-CHA loaded with recombinant human <em>bone</em> <em>morphogenetic</em> <em>protein</em> 2 (BMP, 2 or <em>10</em> microg/block) was implanted with or without vascular insertion. At 3 weeks, IP-CHA/BMP (<em>10</em> microg) composite with vascular insertion exhibited abundant new <em>bone</em> formation in the pores of the deep portion close to the inserted vessels. In contrast, IP-CHA/BMP (<em>10</em> microg) without vascular insertion showed poor <em>bone</em> formation. Histomorphometric analysis demonstrated that vascular insertion significantly increased new <em>bone</em> formation. In IP-CHAs with a lower dose of BMP (2 microg), no <em>bone</em> formation was found, with or without vascular insertion. These results suggest that the present system of integrating a vascular network with IP-CHA is a useful technique for <em>bone</em> tissue engineering.

Reconstruction of large <em>bone</em> defects is limited by insufficient vascularization and slow <em>bone</em> regeneration. The objective of this work was to investigate the effect of spatial and temporal release of recombinant human <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 (BMP2) and vascular endothelial growth factor (VEGF) on the extent of osteogenic and vasculogenic differentiation of human mesenchymal stem cells (hMSCs) and endothelial colony-forming cells (ECFCs) encapsulated in a patterned hydrogel. Nanogels (NGs) based on polyethylene glycol (PEG) macromers chain-extended with short lactide (L) and glycolide (G) segments were used for grafting and timed-release of BMP2 and VEGF. NGs with 12kDa PEG molecular weight (MW), 24 LG segment length, and 60/40L/G ratio (P12-II, NG(<em>10</em>)) released the grafted VEGF in <em>10</em>days. NGs with 8kDa PEG MW, 26 LG segment length, and 60/40L/G ratio (P8-I, NG(21)) released the grafted BMP2 in 21days. hMSCs and NG-BMP2 were encapsulated in a patterned matrix based on acrylate-functionalized lactide-chain-extended star polyethylene glycol (SPELA) hydrogel and microchannel patterns filled with a suspension of hMSCs+ECFCs and NG-VEGF in a crosslinked gelatin methacryloyl (GelMA) hydrogel. Groups included patterned constructs without BMP2/VEGF (None), with directly added BMP2/VEGF, and NG-BMP2/NG-VEGF. Based on the results, timed-release of VEGF in the microchannels in <em>10</em>days from NG(<em>10</em>) and BMP2 in the matrix in 21days from NG(21) resulted in highest extent of osteogenic and vasculogenic differentiation of the encapsulated hMSCs and ECFCs compared to direct addition of VEGF and BMP2. Further, timed-release of VEGF from NG(<em>10</em>) in hMSC+ECFC encapsulating microchannels and BMP2 from NG(21) in hMSC encapsulating matrix sharply increased bFGF expression in the patterned constructs. The results suggest that mineralization and vascularization are coupled by localized secretion of paracrine signaling factors by the differentiating hMSCs and ECFCs.

Primary pulmonary hypertension (PPH) is a potentially lethal disorder, in which heterozygous mutations within the <em>bone</em> <em>morphogenetic</em> <em>protein</em> type II receptor (BMPR2) gene (BMPR2) have been identified. We conducted a molecular study of BMPR2 mutations in 4 Japanese families with familial PPH and 30 Japanese patients with sporadic PPH, and found 13 different mutations, of which <em>10</em> were novel, including missense (n=2), nonsense (n=4), frameshift (n=3), and splice-donor site (n=1) mutations. In total, BMPR2 mutations were found in all 4 familial PPH cases and 12 (40%) of the sporadic PPH cases. Further, a majority of the mutations found were predicted to cause premature termination, as previously reported. In the 9 mutations found in the sporadic cases, 2 were shown to be de novo, 2 were shared in multiple cases, 1 was shared with an FPPH case, and 1 was the same as previously reported in Caucasian FPPH. These results indicate that a substantial portion of Japanese PPH patients carry BMPR2 mutations with considerable heterogeneity.

BACKGROUND

The objective of this study was to evaluate local bone formation at titanium porous oxide (TPO) implant surfaces adsorbed with recombinant human bone morphogenetic protein-2 (rhBMP-2).

METHODS

In vitro studies were used to estimate the kinetics of I125-labeled rhBMP-2 released from TPO surfaces with narrow (N) or open (O) pores. Machined/turned titanium (MT) surfaces served as control. The rat ectopic model was used to assess local bone formation. Briefly, TPO-N, TPO-O, and MT disc implants adsorbed with 5, 10, or 20 microg rhBMP-2, respectively, were implanted subcutaneously into the ventral thoracic region in 5-week-old male Long Evans rats. The animals were euthanized at day 14 postsurgery when implants with surrounding tissues were removed, radiographed, and gross observations recorded. The specimens were processed for histologic evaluation using conventional cut-and-grind techniques. TPO implants without rhBMP-2 included in a preliminary evaluation revealed no evidence of bone formation, tissue encapsulation, or vascularity, thus such controls were not further used.

RESULTS

TPO and MT implant surfaces adsorbed with 5 microg rhBMP-2 retained 2.3-5.4% rhBMP-2 following immersion and rinse in buffer, and 1.1-2.2% rhBMP-2 following repeated immersions and rinses over 27 days. TPO implants retained the most rhBMP-2 and MT implants retained the least. Explants revealed increased hard tissue formation, tissue encapsulation, and vascularity at TPO compared with MT implants. Radiographic observations were consistent with the explant observations. The histologic analysis showed greater amounts of bone formation, osteoblastic cells, osteoid, marrow, tissue encapsulation, vascularity, and bone voids for implants adsorbed with 10 and 20 microg rhBMP-2, and for TPO implants at the 5-microg rhBMP-2 dose. The histometric analysis revealed significantly greater bone formation at TPO-O than at MT implants at the 5-microg rhBMP-2 dose. All surfaces showed significant bone formation at the 10- and 20-microg dose.

CONCLUSIONS

rhBMP-2 adsorbed onto TPO implant surfaces executes an osteoinductive effect including bone contacting the implant surface. This effect is surface- and dose-dependent; the TPO-O surface yielding the most bone at the low discriminating rhBMP-2 dose.

BACKGROUND

Use of recombinant human bone morphogenetic protein-2 (rhBMP-2) has increased considerably since its introduction in 2002. The complications associated with high-dose rhBMP-2 (≥ 40 mg) are unknown. The purpose of our study was to determine outcomes and medical and surgical complications associated with high-dose rhBMP-2 at short-term and long-term follow-up evaluations.

METHODS

Five hundred and two consecutive adult patients who had received high-dose rhBMP-2 as a part of spinal surgery from 2002 to 2009 at one institution were enrolled. Data were entered prospectively and studied and analyzed retrospectively. Surgical procedures in the thoracic and lumbar spine were included. Major and minor complications were documented intraoperatively, perioperatively, and at the latest follow-up examination. Complications potentially associated with rhBMP-2 use were evaluated for correlation with rhBMP-2 dose. Scoliosis Research Society (SRS) and Oswestry Disability Index (ODI) outcome measures were obtained before and after surgery.

RESULTS

On average, 115 mg (range, 40 to 351 mg) of rhBMP-2 was used. The average age of the patients (410 women and ninety-two men) at the time of the index procedure was 52.4 years (range, eighteen to eighty years). There were 265 primary and 237 revision procedures, and 261 patients had interbody fusion. An average of 11.5 vertebrae were instrumented. The average duration of follow-up was forty-two months (range, fourteen to ninety-two months). The diagnoses included idiopathic scoliosis (41%), degenerative scoliosis (31%), fixed sagittal imbalance (18%), and other diagnoses (10%). The rate of intraoperative complications was 8.2%. The rate of perioperative major surgical complications was 11.6%. The rate of perioperative major medical complications was 11.6%. Minor medical complications occurred in 18.9% of the cases, and minor surgical complications occurred in 2.6%. Logistic regression analysis and Pearson correlation did not identify a significant correlation between rhBMP-2 dosage and radiculopathy (r = -0.006), seroma (r = -0.003), or cancer (r = -0.05). Significant improvements in the ODI score (from a mean of 41 points to a mean of 26 points; p < 0.001) and the SRS total score (from a mean of 3.0 points to a mean of 3.7 points; p < 0.001) were noted at the latest follow-up evaluation.

CONCLUSIONS

This is the largest study of which we are aware that examines complications associated with high-dose rhBMP-2. Major surgical complications occurred in 11.6% of patients, and 11.6% experienced major medical complications. There was a cancer prevalence of 3.4%, but no correlation between increasing rhBMP-2 dosage and cancer, radiculopathy (seen in 1% of the patients), or seroma (seen in 0.6%) was found.

OBJECTIVE

To examine the ability of cartilage-like tissue, generated ectopically in a diffusion chamber using recombinant human bone morphogenetic protein 2 (rHuBMP-2), to repair cartilage defects in rats.

METHODS

Muscle-derived mesenchymal cells were prepared by dissecting thigh muscles of 19-day postcoital rat embryos. Cells were propagated in vitro in monolayer culture for 10 days and packed within diffusion chambers (10(6)/chamber) together with type I collagen (CI) and 0, 1, or 10 microg rHuBMP-2, and implanted into abdominal subfascial pockets of adult rats. Tissue pellets were harvested from the diffusion chambers at 2 days to 6 weeks after implantation, and examined by histology, by reverse transcription-polymerase chain reaction (PCR) for aggrecan, CII, CIX, CX, and CXI, MyoD1, and core binding factor a1/runt-related gene 2, and by real-time PCR for CII. Tissue pellets generated in the chamber 5 weeks after implantation were transplanted into a full-thickness cartilage defect made in the patellar groove of the same strain of adult rat.

RESULTS

In the presence of 10 microg rHuBMP-2, muscle-derived mesenchymal cells expressed CII messenger RNA at 4 days after transplantation, and a mature cartilage mass was formed 5 weeks after transplantation in the diffusion chamber. Cartilage was not formed in the presence of 1 microg rHuBMP-2 or in the absence of rHuBMP-2. Defects receiving cartilage engineered with 10 microg rHuBMP-2 were repaired and restored to normal morphologic condition within 6 months after transplantation.

CONCLUSIONS

This method of tissue engineering for repair of articular defects may preclude the need to harvest cartilage tissue prior to mosaic arthroplasty or autologous chondrocyte implantation. Further studies in large animals will be necessary to validate this technique for application in clinical practice.