BACKGROUND

Osteogenic protein-1/bone morphogenetic protein-7 (OP-1/BMP-7), a member of the transforming growth factor-beta superfamily, has been shown to prevent kidney damage from ischemia/reperfusion injury in rats. The molecular events involved in OP-1 action on kidney are not yet understood.

METHODS

In this study, we evaluated the biodistribution of (125)I-labeled OP-1 in rat kidneys. Adult rats received a single intravenous injection of 250 microg (125)I-labeled OP-1 per kg body wt, a dose that was effective in protecting kidneys from ischemic injury. Tissue localization, in situ hybridization, and immunostaining with a specific receptor antibody were performed to identify OP-1 cellular targets. Also, isolated plasma membranes from kidney cortex and medulla regions were analyzed to identify and characterize receptor structural components that recognize OP-1.

RESULTS

At 10 and 180 minutes following injection, the relative uptake of (125)I-labeled OP-1 was consistently higher in kidney cortex than in medulla region. Upon autoradiography, kidney tissue sections revealed that OP-1 bound to the convoluted tubule epithelium, glomeruli, and collecting ducts. Moreover, in situ hybridization and immunostaining methods have shown localization of mRNA transcripts and the protein for BMP receptor type II in the cortex and medulla in similar areas as (125)I-labeled OP-1. Bulk membranes (enriched with plasma membranes) isolated from the cortex and medulla regions of kidney each bound specifically to (125)I-OP-1, and the binding of (125)I-labeled OP-1 was inhibited by unlabeled OP-1 in a dose-dependent manner. However, platelet-derived growth factor, transforming growth factor-beta, insulin-like growth factor, fibroblast growth factors, and other members of BMP family such as BMP-2 and cartilage-derived morphogenetic protein-1/growth and differentiation factor-5 (CDMP-1/GDF-5) failed to inhibit the binding of (125)I-labeled OP-1 to receptors, suggesting a high degree of specificity with which OP-1 bound to kidney receptors. Scatchard analysis of quantitative binding data indicated that the OP-1 receptors of kidney contained a single class of high-affinity binding sites for OP-1 with an association constant (Ka) of 2.26 x 109 mol/L-1 and a binding capacity of 1.01 pmol of OP-1 per mg membrane protein. When analyzed by a ligand blot technique, plasma membranes isolated from kidney cortex and medulla each showed the presence of a prominent specific band with a relative molecular mass (Mr) of 100 kD. Further analysis by Western blotting indicated that an antibody raised against BMP type II receptor effectively recognized the 100 kD OP-1 binding component of kidney plasma membranes.

CONCLUSIONS

We demonstrated, to our knowledge for the first time, the presence of membrane-bound, specific, high-affinity OP-1 receptors in rat kidney tissues, which are likely to mediate OP-1 actions in the kidney. The major OP-1-binding component of the kidney appears to be a long form of BMP type II receptor with a Mr of 100 kD. In vivo and in vitro evidence suggests that the cellular targets for OP-1 are convoluted tubule epithelium, glomeruli, and collecting ducts. OP-1 does not share receptor binding properties with other growth factors, including BMP-2 and CDMP-1, suggesting that its mode of action in kidney appears to be specific.

Post-natal osteogenesis after mechanical trauma or stimulus occurs through either endochondral healing, intramembranous healing or lamellar <em>bone</em> formation. <em>Bone</em> <em>morphogenetic</em> <em>protein</em> 2 (BMP2) is up-regulated in each of these osteogenic processes and is expressed by a variety of cells including osteoblasts and vascular cells. It is known that genetic knockout of Bmp2 in all cells or in osteo-chondroprogenitor cells completely abrogates endochondral healing after full fracture. However, the importance of BMP2 from differentiated osteoblasts and endothelial cells is not known. Moreover, the importance of BMP2 in non-endochondral <em>bone</em> formation such as intramembranous healing or lamellar <em>bone</em> formation is not known. Using inducible and tissue-specific Cre-lox mediated targeting of Bmp2 in adult (<em>10</em>-24 week old) mice, we assessed the role of BMP2 expression globally, by osteoblasts, and by vascular endothelial cells in endochondral healing, intramembranous healing and lamellar <em>bone</em> formation. These three osteogenic processes were modeled using full femur fracture, ulnar stress fracture, and ulnar non-damaging cyclic loading, respectively. Our results confirmed the requirement of BMP2 for endochondral fracture healing, as mice in which Bmp2 was knocked out in all cells prior to fracture failed to form a callus. Targeted deletion of Bmp2 in osteoblasts (osterix-expressing) or vascular endothelial cells (vascular endothelial cadherin-expressing) did not impact fracture healing in any way. Regarding non-endochondral <em>bone</em> formation, we found that BMP2 is largely dispensable for intramembranous <em>bone</em> formation after stress fracture and also not required for lamellar <em>bone</em> formation induced by mechanical loading. Taken together our results indicate that osteoblasts and endothelial cells are not a critical source of BMP2 in endochondral fracture healing, and that non-endochondral <em>bone</em> formation in the adult mouse is not as critically dependent on BMP2.

In order to investigate the osteoinductive properties of allograft used in impaction grafting and the effect of strain during impaction on these properties, we designed an in vitro experiment to measure strain-related release of <em>bone</em> <em>morphogenetic</em> <em>protein</em>-7 (BMP-7) from fresh-frozen femoral head allograft. A total of 40 <em>10</em> mm cubes of cancellous <em>bone</em> were cut from ten samples of fresh-frozen femoral head. The marrow was removed from the cubes and the baseline concentrations of BMP-7 were measured. Specimens from each femoral head were allocated to four groups and subjected to different compressive strains with a material testing machine, after which BMP-7 activity was reassessed. It was present in all groups. There was a linear increase of <em>10</em>2.1 pg/g (95% confidence interval 68.6 to 135.6) BMP-7 for each <em>10</em>% increase in strain. At 80% strain the mean concentration of BMP-7 released (830.3 pg/g <em>bone</em>) was approximately four times that released at 20% strain. Activity of BMP-7 in fresh-frozen allograft has not previously been demonstrated. This study shows that the freezing and storage of femoral heads allows some maintenance of biological activity, and that impaction grafting provides a source of osteoinductive <em>bone</em> for remodelling. We have shown that BMP-7 is released from fresh-frozen femoral head cancellous <em>bone</em> in proportion to the strain applied to the <em>bone</em>. This suggests that the impaction process itself may contribute to the biological process of remodelling and bony incorporation.

BACKGROUND

Formation of blood vessels from pre-existing ones, also termed angiogenesis, is of crucial importance for the outgrowth of tumours beyond 1 - 2 mm³. Therefore, anti-angiogenic therapies, mainly focussing on inhibition of vascular endothelial growth factor (VEGF) are used in clinical therapy. However, although initially reducing tumour size, therapy resistance occurs frequently and new targets are needed. A possible target is activin receptor-like kinase (ALK)-1, a transforming growth factor (TGF)-β type-I receptor, which binds <em>bone</em> <em>morphogenetic</em> <em>protein</em> (BMP)-9 and -<em>10</em> with high affinity and has an important role in regulating angiogenesis.

METHODS

Several approaches to interfere with ALK1 signalling have been developed, that is, ALK1 neutralising antibodies and a soluble ALK1 extracellular domain/Fc fusion protein (ALK1-Fc), acting as a ligand trap. In this review, we discuss the involvement of ALK1 in angiogenesis, in a variety of diseases and the current status of the development of ALK1 inhibitors for cancer therapy.

CONCLUSIONS

Based on current, mainly preclinical studies on inhibition of ALK1 signalling by ligand traps and neutralising antibodies, targeting ALK1 seems very promising. Both ALK1-Fc and neutralising antibodies strongly inhibit angiogenesis in vitro and in vivo. The results from the first Phase I clinical trials are to be reported soon and multiple Phase II studies are ongoing.

<em>Bone</em> <em>morphogenetic</em> <em>proteins</em> (BMPs) enhance neurite outgrowth in nerve growth factor (NGF)-stimulated PC12 cells. To investigate the mechanism of this potentiating effect, real-time PCR was used to analyze the expression of 45 selected genes. A robust increase in expression of <em>10</em> immediate early genes including Egr1-4, Hes1, Junb, Jun and Fos was observed already after 1 h treatment with NGF alone. NGF plus BMP4 further increased these transcripts at 1 h and activated 18 additional genes. BMP4 alone induced Smad6, Mtap1b and Hes1. Egr3 was the gene most strongly upregulated by NGF and BMP4. However, luciferase assays showed that the cloned Egr3 proximal promoter was not involved in the BMP4 potentiation. Blocking Egr3 and Junb function by dominant-negative constructs reduced neurite outgrowth under stimulating conditions, proving that activation of members of both the Egr and Jun families is necessary for maximal PC12 cell response to NGF and BMP4.

BACKGROUND

Conventional oral/maxillofacial implants reach osseointegration over several months during which the titanium fixtures interact with alveolar bone. The objective of this study was to determine if adsorbing recombinant human bone morphogenetic protein-2 (rhBMP-2) onto a titanium porous oxide (TPO) implant surface might enhance or accelerate local bone formation and support osseointegration in a large animal oral/maxillofacial orthotopic model.

METHODS

Endosseous implants with a TPO surface were installed into the edentulated posterior mandible in eight adult Hound Labrador mongrel dogs. The implant surface had been adsorbed with rhBMP-2 at 0.2 or 4.0 mg/ml. TPO implants without rhBMP-2 served as control. Treatments were randomized between jaw quadrants. Mucosal flaps were advanced and sutured leaving the implants submerged. Clinical and radiographic evaluations were made immediately post-surgery, at day 10 (suture removal), and week 4 and 8 post-surgery. The animals received fluorescent bone markers at week 3, 4, and at week 8 post-surgery, when they were euthanized for histologic analysis.

RESULTS

TPO implants coated with rhBMP-2 exhibited dose-dependent bone remodelling including immediate resorption and formation of implant adjacent bone, and early establishment of clinically relevant osseointegration. The resulting bone-implant contact, although clinically respectable, appeared significantly lower for rhBMP-2-coated implants compared with the control [rhBMP-2 (0.2 mg/ml) 43.3+/-10.8%versus 71.7+/-7.8%, p<0.02; rhBMP-2 (4.0 mg/ml) 35.4+/-10.6%versus 68.2+/-11.0%, p<0.03].

CONCLUSIONS

rhBMP-2 adsorbed onto TPO implant surfaces initiates dose-dependent peri-implant bone re-modelling resulting in the formation of normal, physiologic bone and clinically relevant osseointegration within 8 weeks.

METHODS

Retrospective database analysis.

OBJECTIVE

A nationwide population-based database was analyzed to assess the utilization trends of bone morphogenetic protein (BMP) in spine fusion surgery from 2002-2011.

BACKGROUND

The utilization of off-label BMP in spine procedures is not well characterized. The purpose of this study was to analyze a population-based database to characterize the national trends of BMP utilization in terms of incidence, demographics, costs, and mortality.

METHODS

Data from the Nationwide Inpatient Sample of the Healthcare Cost and Utilization Project was queried for each year from 2002-2011. Patients undergoing an anterior cervical fusion or posterior cervical fusion, anterior lumbar fusion or posterior lumbar fusion, or a posterior thoracic fusion were identified and separated into cohorts. The frequency of BMP utilization was assessed in each surgical cohort by year. Patient demographics, hospital parameters, costs, and mortality rates were assessed.

RESULTS

The adjusted annual number of procedures with BMP increased from 1116 in 2002 to 79,294 in 2011 (P < 0.001), representing 26.9% of all spinal fusion procedures. The rate of BMP utilization within each surgical cohort also significantly increased during the 10-year period (P < 0.001). The posterior lumbar fusion cohort accounted for the majority of spinal fusions that used BMP, representing 76.8% of all spinal fusions between 2002 and 2011. The anterior lumbar fusion cohort was associated with the highest proportion of BMP utilization, peaking at 56.9% of all anterior lumbar interbody fusions in 2006. The trend of BMP utilization in the anterior cervical fusion cohort peaked in 2007 with 10.6% of cases and then declined to 6.4% in 2011. There was a statistically significant trend of older patients with increasing comorbidities receiving BMP during this period. Hospital costs (adjusted for inflation) significantly increased an average of $9560 from 2002-2010. There were no significant trends with regard to the length of hospitalization stay and mortality rates during this period.

CONCLUSIONS

This nonconflicted study demonstrates that the utilization of BMP has dramatically increased from 2002-2011. Interestingly, off-label application of BMP accounts for the vast majority of BMP utilization. The increase in the total hospital costs is likely multifactorial; older patients with more comorbidities undergoing surgery as well as the increasing utilization of BMP are all likely contributory factors. The length of hospitalization stay and mortality rates did not increase during the 10-year period.

A balance between cell survival and apoptosis is essential for animal development. Although proper development involves multiple interactions between germ layers, little is known about the intercellular and intertissue signaling pathways that promote cell survival in neighboring or distant germ layers. We found that serum- and glucocorticoid-inducible kinase 1 (SGK1) promoted ectodermal cell survival during early Xenopus embryogenesis through a non-cell-autonomous mechanism. Dorsal depletion of SGK1 in Xenopus embryos resulted in shortened axes and reduced head structures with defective eyes, and ventral depletion led to defective tail morphologies. Although the gene encoding SGK1 was mainly expressed in the endoderm and dorsal mesoderm, knockdown of SGK1 caused excessive apoptosis in the ectoderm. SGK1-depleted ectodermal explants showed little or no apoptosis, suggesting non-cell-autonomous effects of SGK1 on ectodermal cells. Microarray analysis revealed that SGK1 knockdown increased the expression of genes encoding FADD (Fas-associated death domain <em>protein</em>) and caspase-<em>10</em>, components of the death-inducing signaling complex (DISC). Inhibition of DISC function suppressed excessive apoptosis in SGK1-knockdown embryos. SGK1 acted through the transcription factor nuclear factor κB (NF-κB) to stimulate production of <em>bone</em> <em>morphogenetic</em> <em>protein</em> 7 (BMP7), and overexpression of BMP7 in SGK1-knockdown embryos reduced the abundance of DISC components. We show that phosphoinositide 3-kinase (PI3K) functioned upstream of SGK1, thus revealing an endodermal and mesodermal pathway from PI3K to SGK1 to NF-κB that produces BMP7, which promotes ectodermal survival by decreasing DISC function.

Human mesenchymal stem cells obtained from the iliac crest of a single donor were investigated for cell proliferation, cell cycle profile, gene expression, and ultrastructural changes using electron microscopy. The human mesenchymal stem cells significantly increased their cell number by day 2 after treatment with <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 alone, or basic fibroblast growth factor alone or combinations of both <em>proteins</em> under serum-free conditions (p < 0.01). The human mesenchymal stem cells showed marked expression of cell nuclear antigen, notably at day 1, and pituitary tumor transforming gene throughout the experiment, suggesting cell cycle progression by <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 treatment. In addition, strong cellular nuclear bromodeoxyuridine incorporation was seen by immunocytochemistry. Fluorescence-activated cell sorting also showed a similar pattern of cell cycle progression with <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 treatment in serum-free medium and <em>10</em>% fetal bovine serum treatment. The <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2-treated human mesenchymal stem cells showed heterochromatin in the nucleus, suggesting cell differentiation, and well-developed granular endoplasmic reticulum, indicative of <em>protein</em> production. Overall, the human mesenchymal stem cells successfully proliferated with appropriate cell cycle progression and the cell ultrastructural morphology suggested marked nuclear and granular endoplasmic reticulum induction by <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 treatment in serum-free medium.

Traditional bone grafting relies upon the incorporation of a bone-cell bearing structure into a recipient site. The graft serves as a scaffold that is eventually replaced and remodeled. This process is known as osteoconduction. Recombinant human bone morphogenetic protein-2 (rhBMP-2) is commercially available as an acellular implant in which the protein is bound to an absorbable collagen sponge (ACS). The rhBMP-2/ACS implant converts undifferentiated mesenchymal stem cells into osteoblasts and promotes an intense local neovascular response. This process, known as osteoinduction, produces bone via membranous, chondroid, or endochondral ossification. The type of bone synthesis depends upon the mesenchymal substrate and the local cellular environment. Using this simple technique, bone defects can be resynthesized with good outcomes and a significant reduction in donor site morbidity. Repair of a critical-sized mandibular resection defect with ISO is described. Basic science concepts of rhBMP-2, relevant histopathologic findings, and clinical application are described.

The present experimental study sought to determine the effect of high-dose irradiation on the rat mandible in order to establish an experimental model of radiogenic <em>bone</em> damage. The left mandibles of 20 adult Wistar rats were irradiated (single fraction 1500cGy, total dose 60Gy) by means of a hypofractionated stereotactic radiotherapy (hfSRT) over a period of 6 weeks. Follow-up was 6 weeks (group 1, n=<em>10</em>) and 12 weeks (group 2, n=<em>10</em>). The contralateral mandibles as well as 5 non-irradiated animals served as controls. Primary endpoints were fibrosis, loss of cell count, decreased immunohistochemical labelling for <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 (BMP-2) and osteocalcin as well as increased expression of transforming growth factor (TGF-beta). Cell loss, progressive fibrosis, and focal necrosis were detected in all irradiated sites. Quantitative measurement revealed 32.0+/-8.7% and 37.3+/-9.5% empty osteocyte lacunae for groups 1 and 2 resp., compared to 16.3+/-4.7% and 18.9+/-4.9% on the contralateral side and 7.9+/-1.7% for unirradiated controls (Mann-Whitney U test; p<.01). BMP-2 and osteocalcin labelling showed a marked decrease in irradiated and contralateral sides while TGF-beta was expressed strongly in irradiated sites only (for all p<.05). External hypofractionated irradiation with a total dose of 60Gy is feasible in rats and yields all histologic changes attributed to osteoradionecrosis (ORN) after a follow-up of 6 weeks. The irradiation protocol is suitable for an assessment of regenerative options in severe radiogenic <em>bone</em> damage. As a split mouth design entails major inaccuracies healthy animals have to be used as controls.

Monocytes have the potential to differentiate to either macrophages, dendritic cells, or to osteoclasts. The microenvironment, particularly cytokines, directs the monocyte differentiation. Receptors of NFκB (RANK) ligand, tumor necrosis factor (TNF) α, or interleukin- (IL-) 8 have be identified as inducers of osteoclastogenesis, whereas others, such as IL-<em>10</em> or transforming growth factor (TGF)ß inhibit osteoclast generation or induce differentiation towards a dendritic cell type. We now describe that <em>bone</em> <em>morphogenetic</em> <em>protein</em> (BMP) 7/osteogenic <em>protein</em>- (OP-) 1 inhibited the differentiation of human CD14+ monocytes to osteoclasts. In the presence of BMP7/OP-1 the transcription factors c-Fos and NFATc1, though upregulated and translocated to the nucleus in response to either RANKL or IL-8, did not persist. In parallel, MafB, a transcription factor expressed by monocytes and required for differentiation to macrophages but inhibiting osteoclast generation, was preserved. Because both persistence of NFATc1 and downregulation of MafB are crucial for osteoclastogenesis, we conclude that BMP7/OP-1 inhibits the generation of osteoclasts by interfering with signalling pathways.

OBJECTIVE

To systematically assess the literature regarding the clinical, histological, and radiographic outcome of bone morphogenetic proteins (BMP-2, BMP-7), growth/differentiation factor-5 (GDF-5), platelet-derived growth factor (PDGF), and parathyroid hormone (PTH) for localized alveolar ridge augmentation.

METHODS

Five separate Medline searches were performed in duplicate for human and animal studies, respectively. The primary outcome of the included studies was bone regeneration of localized alveolar ridge defects or craniofacial defects.

RESULTS

In six human studies, BMP-2 affected local bone augmentation with increasing volume for higher doses. A majority (43 of 45) of animal studies using BMP-2 showed a positive effect in favour of the growth factor (GF). In six of eight studies, a positive effect was associated with the use of BMP-7. Only one animal study was included for GDF-5 revealing statistically significantly higher bone volume. Regarding PDGF, statistically significantly higher bone volume was observed in five of 10 included studies. Four animal studies using PTH revealed statistically significantly more bone regeneration compared with controls.

CONCLUSIONS

Differing levels and quantity of evidence were noted to be available for the GFs evaluated, revealing that BMP-2, BMP-7, GDF-5, PDGF, and PTH may stimulate local bone augmentation to various degrees. Human data for the potential of rhBMP-2 are supportive.

Smads are cytoplasmic signal transducers of transforming growth factor-beta (TGF-beta) and <em>bone</em> <em>morphogenetic</em> <em>proteins</em> (BMPs). Their relation to fracture healing is unknown. This study examined the temporal <em>protein</em> expression of Smads, together with TGF-beta and BMPs, using immunohistochemistry in a rodent fracture model. Over-expression of TGF-beta, BMPs-2, 4, and 7, common-mediator Smad (Smad4), and receptor-regulated Smads (Smads1, 2, 3, and 5) versus lower levels of inhibitory Smad (Smad6), were detected at day 3 in osteogenic cells in the thickened periosteum and <em>bone</em> marrow at the fracture sites. At day <em>10</em>, Smad6 increased dramatically, Smad2, Smad3, and Smad4 remained elevated while Smad1 and Smad5 decreased in the fracture callus. Smad7 was expressed only in vascular endothelial cells. By day 28, when new <em>bone</em> had replaced the fracture callus, all the <em>protein</em> regulators decreased, approaching control levels. During fracture healing, the expression patterns of Smads1 and 5 were similar to that of BMPs-2 and 7 whereas the expression of Smads2 and 3 was parallel with that of TGF-beta. The Smad family, associated with BMPs and TGF-beta, may play an important role in the early stage of rat fracture healing.

BACKGROUND

Reconstruction of the pediatric calvaria is frequently complicated by a shortage of <em>bone</em>. This problem is most apparent between 2 and <em>10</em> years of age, when the osteogenic potential of the dura is diminished and the diploic space has not matured to the point that split-thickness calvarial grafting is practical. In this article, the authors evaluate and compare the relative efficacy of adipose-derived stem cells, <em>bone</em> <em>morphogenetic</em> <em>protein</em> (BMP)-2, and adipose-derived stem cells osteoinduced with BMP-2 in addressing these defects.

METHODS

Cranial defects measuring 15×15 mm were created in New Zealand White rabbits. Five treatment modalities were compared: no repair (surgical control); untreated acellular collagen sponge (vehicle control); BMP-2 on acellular collagen sponge; adipose-derived stem cells on acellular collagen sponge; and osteoinduced adipose-derived stem cells on acellular collagen sponge. Osteogenesis was assessed with radiology and histology. Statistical significance was determined by analysis of variance.

RESULTS

No significant difference in osseous healing was observed among empty controls (32.8 percent), acellular collagen sponge alone (34.4 percent), adipose-derived stem cells on acellular collagen sponge (33.9 percent), and osteoinduced adipose-derived stem cells on acellular collagen sponge (40.2 percent). Defects reconstructed with recombinant human BMP-2/acellular collagen sponge were on average 96.9 percent ossified, significantly (p<0.05) more than the defects in all other groups.

CONCLUSIONS

BMP-2-based tissue engineering is a viable approach to craniofacial reconstruction. Adipose-derived stem cells did not significantly augment this process as modeled here. Advances in the understanding of craniofacial biology, and of protein- and cell-based therapies, will enhance the efficacy of tissue-engineering strategies for this problem in the future.

Congenital pseudarthrosis of the tibia is an uncommon manifestation of neurofibromatosis type 1 (NF1), but one that remains difficult to treat due to anabolic deficiency and catabolic excess. <em>Bone</em> grafting and more recently recombinant human <em>bone</em> <em>morphogenetic</em> <em>proteins</em> (rhBMPs) have been identified as pro-anabolic stimuli with the potential to improve the outcome after surgery. As an additional pharmaceutical intervention, we describe the combined use of rhBMP-2 and the bisphosphonate zoledronic acid in a mouse model of NF1-deficient fracture repair. Fractures were generated in the distal tibiae of neurofibromatosis type 1-deficient (Nf1(+/-)) mice and control mice. Fractures were open and featured periosteal stripping. All mice received <em>10</em> μg rhBMP-2 delivered in a carboxymethylcellulose carrier around the fracture as an anabolic stimulus. Bisphosphonate-treated mice also received five doses of 0.02 mg/kg zoledronic acid given by intraperitoneal injection. When only rhBMP but no zoledronic acid was used to promote repair, 75% of fractures in Nf1(+/-) mice remained ununited at three weeks compared with 7% of controls (p < 0.001). Systemic post-operative administration of zoledronic acid halved the rate of ununited fractures to 37.5% (p < 0.07). These data support the concept that preventing <em>bone</em> loss in combination with anabolic stimulation may improve the outcome following surgical treatment for children with congenital pseudarthosis of the tibia and NF1.

OBJECTIVE

Open fractures with bone loss are common, disabling injuries. Biodegradable, load-bearing scaffolds able to carry high concentrations of local antibiotics are an emerging technology to address these injuries. This study investigates the use of such scaffolds with gentamicin (along with bone morphogenetic protein) in an infected rat open fracture model to decrease osteomyelitis and promote fracture healing.

METHODS

A contaminated open fracture was created in 32 Brown Norway rats. A comminuted femoral fracture was created, followed by crushing, and the 5-mm bone defect was inoculated with Staphylococcus aureus (10 colony-forming units/mL) and Escherichia coli (10 colony-forming units/mL). The scaffold was stabilized in the defect with an intramedullary Kirschner wire. Gentamicin was loaded onto the scaffolds at two doses, either 10 mg (n = 12) or 20 mg (n = 10). Controls (n = 10) received no antibiotics. All three groups had 10 microg bone morphogenetic protein loaded on the scaffold. Serial radiographs were obtained. Microbiologic analysis, microcomputed tomography, and histology were performed.

RESULTS

There was a statistically significant difference in the radiographic evidence of osteomyelitis (P = 0.004) and callus formation (P = 0.021) between the treated and control groups. Bone culture analysis results were not significant for S. aureus (P = 0.29) or E. coli (P = 0.25). There was no difference in the mean scaffold volume or density of the three treatment groups.

CONCLUSIONS

Our results suggest that gentamicin applied to a biodegradable scaffold is effective at decreasing radiographically defined osteomyelitis in an infected open fracture.

Basement membrane plays important roles in hair growth. We characterized changes in laminin isoform expression during hair cycling. At the mRNA level, laminin-511 (<em>10</em>) expression underwent a steady increase during anagen stages. In contrast, laminin-332 (5) expression was initially upregulated in outer root sheath (ORS) keratinocytes at anagen II and then transiently downregulated. Laminin-332 significantly increased coincident with the signal in inner root sheath and hair matrix cells after anagen IV. Levels of laminin-332 <em>proteins</em> were also upregulated at late anagen I-III but dropped after anagen IV. This decrease coincided with increased levels of mRNA encoding the two proteases, membrane type 1 metallo<em>protein</em>ase and <em>bone</em> <em>morphogenetic</em> <em>protein</em> 1, involved in laminin-332 processing. Immunohistochemistry demonstrated that laminin-332 and alpha6 beta4 integrin were well colocalized, but their signals were remarkably decreased in the lower half of follicles after anagen VI. Consistent with these data, ultrastructurally mature hemidesmosomes were seen in ORS keratinocytes at anagen II, whereas at anagen VI, only fragmental hemidesmosomes were present. In hair follicle culture, laminin-511 (<em>10</em>)/521 (11)-rich human placental laminin enhanced hair growth, whereas recombinant laminin-332 antagonized hair growth induced by laminin-511. Our results indicate a positive role for laminin-511 and a negative role for laminin-332 on hair growth.

During lung development, signaling by Fgf<em>10</em> (fibroblast growth factor <em>10</em>) and its receptor Fgfr2b is critical for induction of a gene network that controls proliferation, differentiation, and branching of the epithelial tubules. The downstream events triggered by Fgf<em>10</em>-Fgfr2b signaling during this process are still poorly understood. In a global screen for transcriptional targets of Fgf<em>10</em>, we identified Ctsh (cathepsin H), a gene encoding a lysosomal cysteine protease of the papain family, highly up-regulated in the developing lung epithelium. Here we show that among other cathepsin genes present in the lung, Ctsh is the only family member selectively induced by Fgf<em>10</em> in the lung epithelium. We provide evidence that, during branching morphogenesis, epithelial expression of Ctsh overlaps temporally and spatially with that of Bmp4 (<em>bone</em> <em>morphogenetic</em> <em>protein</em> 4), another target of Fgf<em>10</em>. Moreover, we show that Ctsh controls the availability of mature Bmp4 <em>protein</em> in the embryonic lung and that inhibiting Ctsh activity leads to a marked accumulation of Bmp4 <em>protein</em> and disruption of branching morphogenesis. Tightly controlled levels of Bmp4 signaling are critical for patterning of the distal lung epithelium. Our study suggests a potentially novel posttranscriptional mechanism in which Ctsh rapidly removes Bmp4 from forming buds to limit Bmp4 action. The presence of both Ctsh and Bmp4 or Bmp4 signaling activity in other developing structures, such as the kidney, yolk sac, and choroid plexus, suggests a possible general role of Ctsh in regulating Bmp4 proteolysis in different <em>morphogenetic</em> events.

BACKGROUND

Tissue-engineering techniques combined with gene therapy have been recently reported to improve osteogenesis. In this study, tissue-engineered bone constructed by human Bone Morphogenetic Protein 4 (hBMP-4) gene-modified bone marrow stromal cells (bMSCs) was explored in an ectopic bone formation model in rabbits.

METHODS

A pEGFP-hBMP-4 mammalian plasmid (EGFP: Enhanced Green Fluorescent Protein) was constructed by subcloning techniques. bMSCs obtained from rabbits were cultured and transfected with either pEGFP-hBMP-4, pEGFP or left uninfected in vitro. Transfer efficiency was detected through the expression of EGFP. Transcription of the target gene was detected by RT-PCR. Alkaline phosphatase (ALP) and Von Kossa tests were also conducted to explore the phenotypes of osteoblasts. The autologous bMSCs of the 3 groups were then combined with Natural Non-organic Bone (NNB), a porous hydroxyapatite implant with a dimension of 6 mm x 6 mm x 3 mm, at a concentration of 5 x 10(7) cells/ml. They were subsequently implanted into 6 rabbits subcutaneously using NNB alone as a blank control (6 implants per group). Four weeks after surgery, the implants were evaluated with histological staining and computerized analysis of new bone formation.

RESULTS

pEGFP-hBMP-4 expression plasmid was constructed. Under optimal conditions, gene transfer efficiency reached more than 30%. Target gene transfer could strengthen the transcription of BMP-4, and increase the expression of ALP as well as the number of calcium nodules. In the ectopic animal model, NNB alone could not induce new bone formation. The new bone area formed in the bMSCs group was (17.2 +/- 7.1)%, and pEGFP group was (14.7 +/- 6.1)%, while pEGFP-hBMP-4 group was (29.5 +/- 8.2)%, which was the highest among the groups (F = 7.295, P < 0.01).

CONCLUSIONS

The mammalian hBMP-4 expression plasmid was successfully constructed and a comparatively high transfer efficiency was achieved. The gene transfer technique enhanced the expression of BMP-4 and promoted differentiation from bMSCs to osteoblasts. These in vivo results suggested that transfection of bMSCs with hBMP-4 might be a suitable method to enhance their inherent osteogenic capacity for bone tissue engineering applications.

Indian hedgehog (Ihh) is highly expressed in prehypertrophic chondrocytes in vivo and has been proposed to regulate the proliferation and maturation of chondrocytes and <em>bone</em> collar formation in the growth plate. In high-density cultures of rabbit growth-plate chondrocytes, Ihh mRNA was also expressed at the highest level in the prehypertrophic stage. To explore endogenous factors that regulate Ihh expression in chondrocytes, we examined the effects of various growth factors on Ihh mRNA expression in this system. Retinoic acid (RA) and <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 enhanced Ihh mRNA expression, whereas PTH/PTH-related peptide (PTHrP) markedly suppressed Ihh expression. RA at more than <em>10</em>(-8) M induced the expression of Ihh and Patched 1 (Ptc1) within 3 h, before it increased the type X collagen mRNA level at 6-24 h. Cycloheximide blocked the up-regulation of Ihh by RA, indicating the requirement of de novo <em>protein</em> synthesis for this stimulation. These findings suggest that RA is involved in the up-regulation of Ihh during endochondral <em>bone</em> formation. In contrast to RA, PTH (1-84) at <em>10</em>(-7) M abolished the mRNA expression of Ihh and Ptc1 within 2-4 h, before it suppressed the expression of type X collagen at 12-24 h. The inhibition of Ihh expression by PTH (1-84) did not require de novo <em>protein</em> synthesis. PTH (1-34), PTHrP (1-34), and (Bu)(2)cAMP also suppressed Ihh expression. On the other hand, Ihh has been reported to induce PTHrP synthesis in the perichondrium. Consequently, the direct inhibitory action of PTH/PTHrP on Ihh appears to be a negative feedback mechanism that prevents excess PTHrP accumulation in cartilage.

The spatial and temporal pattern of the appearance of the fibroblast growth factor <em>proteins</em> (FGF-8 and FGF-<em>10</em>), the <em>bone</em> <em>morphogenetic</em> <em>proteins</em> (BMP-2/4 subfamily and BMP-7) and the vascular endothelial growth factor <em>protein</em> (VEGF) was investigated in the human mesonephros and metanephros of the 5-9 week-old conceptuses. In the mesonephros, both FGF's and BMP's were found in all structures and their expression slightly decreased in the early fetal period. VEGF positivity appeared in all mesonephric structures, and increased in the fetal period coincidently with formation of the mesonephric blood vessel network. In the metanephros, FGF-8 first appeared only in the metanephric mesenchyme, but from the 7th week on, its reactivity increased and spread to other metanephric structures. FGF-<em>10</em> positive cells appeared in all metanephric structures already in the 5th week, and slightly intensified with progression of development. Cell survival and nephrogenesis in the permanent kidney might be associated with the appearance of both growth factors. Both BMP-2/4 and BMP-7 displayed a similar pattern of reactivity in all metanephric structures, and their reactivity intensified with advancing development. Alterations in their pattern of appearance might lead to the formation of small and dysplastic kidneys. Already in the earliest developmental stages, VEGF <em>protein</em> appeared in all metanephric structures. At later stages, VEGF showed more intense reaction in the collecting system than in the differentiating nephrons and interstitium. Due to VEGF involvement in vasculogenesis and angiogenesis, abnormal VEGF appearance might lead to impaired formation of the blood vessel network in the human permanent kidney.

<em>Bone</em> <em>morphogenetic</em> <em>protein</em> family members (BMPs) are essential signaling molecules during limb development and, in this process, fibroblast growth factor family members (FGFs) cooperate with BMPs. FGFs also exert anabolic effects in <em>bone</em> when systemically or locally applied. Thus, it is likely that the cooperation with FGFs also occurs in BMP-induced ectopic <em>bone</em> formation and that the exogenous FGF application would promote this <em>bone</em> formation. In the present study, after subcutaneously implanting recombinant human BMP-2 (rhBMP-2) in rats, we examined the expression of FGF-4 and FGF receptors (FGFRs) mRNAs and the effect of exogenous recombinant human FGF-4 (rhFGF-4) on <em>bone</em> formation. Three days after implantation, the pellets containing rhBMP-2 were surrounded by fibroblastic mesenchymal cells; on day 7, cartilage tissue appeared; on day <em>10</em>, hypertrophic chondrocytes and a small amount of mineralized tissue were observed; and, on day 14, the amount of mineralized tissue increased. Reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that FGF-4 expression appeared at early stages (days 3 and 7) and its expression decreased at later stages (days <em>10</em>, 14, and 21), whereas FGFRs were expressed continuously. In situ hybridization revealed that, on days 3 and 7, FGF-4, and FGFR subtypes 1 and 2 (FGFR-1 and FGFR-2) were expressed in mesenchymal cells and chondrocytes, and in the area of alkaline phosphatase (ALP) expression. On day <em>10</em>, FGF-4 was not detected, whereas the expression of FGFR-1 and FGFR-2 was detectable in the area of alkaline phosphatase (ALP) expression. Injection of rhFGF-4 on days 2, 3, and 4 enhanced the mineralized tissue formation induced by rhBMP-2; however, neither rhFGF-4 treatment on days 6, 7, and 8 nor rhFGF-4 treatment on days 9, <em>10</em>, and 11 influenced the amount of rhBMP-2-induced mineralization. Our results indicate that FGF-4 and FGFR signals play important roles during rhBMP-2-induced <em>bone</em> formation. We further suggest that the combination of rhBMP-2 and rhFGF-4 would be useful for <em>bone</em> augmentation.

Lung development is a highly regulated process directed by mesenchymal-epithelial interactions, which coordinate the temporal and spatial expression of multiple regulatory factors required for proper lung formation. The Iroquois homeobox (Irx) genes have been implicated in the patterning and specification of several Drosophila and vertebrate organs, including the heart. Herein, we investigated whether the Irx genes play a role in lung morphogenesis. We found that Irx1-3 and Irx5 expression was confined to the branching lung epithelium, whereas Irx4 was not expressed in the developing lung. Antisense knockdown of all pulmonary Irx genes together dramatically decreased distal branching morphogenesis and increased distention of the proximal tubules in vitro, which was accompanied by a reduction in surfactant <em>protein</em> C-positive epithelial cells and an increase in beta-tubulin IV and Clara cell secretory <em>protein</em> positive epithelial structures. Transmission electron microscopy confirmed the proximal phenotype of the epithelial structures. Furthermore, antisense Irx knockdown resulted in loss of lung mesenchyme and abnormal smooth muscle cell formation. Expression of fibroblast growth factors (FGF) 1, 7, and <em>10</em>, FGF receptor 2, <em>bone</em> <em>morphogenetic</em> <em>protein</em> 4, and Sonic hedgehog (Shh) were not altered in lung explants treated with antisense Irx oligonucleotides. All four Irx genes were expressed in Shh- and Gli(2)-deficient murine lungs. Collectively, these results suggest that Irx genes are involved in the regulation of proximo-distal morphogenesis of the developing lung but are likely not linked to the FGF, BMP, or Shh signaling pathways.