The in vitro elution of BMP-7 from demineralized bone matrix

Demineralized bone matrix (DBM) grafts induce new bone formation by locally releasing matrix-associated growth factors, such as bone morphogenetic proteins (BMPs), to the surrounding tissue after implantation. However, the release kinetics of BMPs from DBM lack characterization. Such information can potentially help to improve processing techniques to maximize graft osteoinductive potential, as well as increase understanding of the osteoinductive process itself. We produced DBM with three particle size ranges from bovine cortical bone, i.e., <106, 106–300, and 300–710?μm and extracted 1.5?g of each size range in 40?ml of Sorensen’s buffer at room temperature for up to 168?h. The BMP-7 concentration of the DBM and the buffer were measured at each time point using enzyme-linked immunosorbant assay. Based on measurement of the concentration of BMP-7 in the buffer, the 0–8?h elution rate was high, i.e., 3.3, 2.9, and 2.2?ng BMP-7/g DBM?h, and for the 8–168?h interval was much lower, at 0.039, 0.15, and 0.11?ng BMP-7/g DBM?h for the three size ranges, respectively. By 168?h, there was no indication that elution was nearing completion. Measurement of the residual BMP-7 remaining in the DBM as a function of time yielded unexpected results, i.e., after the BMP-7 content of the DBM declined for the first 4–6?h, it paradoxically increased for the remaining interval. We propose a two-compartment model to help explain these results in terms of the possible distribution of BMP-7 in bone matrix.     

Enhancement of ectopic bone formation by bone morphogenetic protein-2 delivery using heparin-conjugated PLGA nanoparticles with transplantation of bone marrow-derived mesenchymal stem cells

This study was performed to determine if a combination of previously undifferentiated bone marrow-derived mesenchymal stem cells (BMMSCs) and exogenous bone morphogenetic protein-2 (BMP-2) delivered via heparin-conjugated PLGA nanoparticles (HCPNs) would extensively regenerate bone in vivo. In vitro testing found that the HCPNs were able to release BMP-2 over a 2-week period. Human BMMSCs cultured in medium containing BMP-2-loaded HCPNs for 2 weeks differentiated toward osteogenic cells expressing alkaline phosphatase (ALP), osteopontin (OPN) and osteocalcin (OCN) mRNA, while cells without BMP-2 expressed only ALP. In vivo testing found that undifferentiated BMMSCs with BMP-2-loaded HCPNs induce far more extensive bone formation than either implantation of BMP-2-loaded HCPNs or osteogenically differentiated BMMSCs. This study demonstrates the feasibility of extensive in vivo bone regeneration by transplantation of undifferentiated BMMSCs and BMP-2 delivery via HCPNs. Sung Eun Kim and Oju Jeon equally contributed to this work     

Effect of guided bone regeneration on bone quality surrounding dental implants

Bone quality as well as its quantity at the implant interface is responsible for determining stability of the implant system. The objective of this study is to examine the nanoindentation based elastic modulus (E) at different bone regions adjacent to titanium dental implants with guided bone regeneration (GBR) treated with DBM and BMP-2 during different post-implantation periods. Six adult male beagle dogs were used to create circumferential defects with buccal bone removal at each implantation site of mandibles. The implant systems were randomly assigned to only GBR (control), GBR with demineralized bone matrix (DBM), and GBR with DBM + recombinant human bone morphogenetic protein-2 (rhBMP-2) (BMP) groups. Three animals were sacrificed at each 4 and 8 weeks of post-implantation healing periods. Following buccolingual dissection, the E values were assessed at the defects (Defect), interfacial bone tissue adjacent to the implant (Interface), and pre-existing bone tissue away from the implant (Pre-existing). The E values of BMP group had significantly higher than control and DBM groups for interface and defect regions at 4 weeks of post-implantation period and for the defect region at 8 weeks (p < 0.043). DBM group had higher E values than control group only for the defect region at 4 weeks (p < 0.001). The current results indicate that treatment of rhBMP-2 with GBR accelerates bone tissue mineralization for longer healing period because the GBR likely facilitates a microenvironment to provide more metabolites with open space of the defect region surrounding the implant.     

Electrospun PLLA nanofiber scaffolds and their use in combination with BMP-2 for reconstruction of bone defects

Introduction

Adequate migration and differentiation of mesenchymal stem cells is essential for regeneration of large bone defects. To achieve this, modern graft materials are becoming increasingly important. Among them, electrospun nanofiber scaffolds are a promising approach, because of their high physical porosity and potential to mimic the extracellular matrix (ECM).

Materials and Methods

The objective of the present study was to examine the impact of electrospun PLLA nanofiber scaffolds on bone formation in vivo, using a critical size rat calvarial defect model. In addition we analyzed whether direct incorporation of bone morphogenetic protein 2 (BMP-2) into nanofibers could enhance the osteoinductivity of the scaffolds. Two critical size calvarial defects (5 mm) were created in the parietal bones of adult male Sprague-Dawley rats. Defects were either (1) left unfilled, or treated with (2) bovine spongiosa, (3) PLLA scaffolds alone or (4) PLLA/BMP-2 scaffolds. Cranial CT-scans were taken at fixed intervals in vivo. Specimens obtained after euthanasia were processed for histology, histomorphometry and immunostaining (Osteocalcin, BMP-2 and Smad5).

Results

PLLA scaffolds were well colonized with cells after implantation, but only showed marginal ossification. PLLA/BMP-2 scaffolds showed much better bone regeneration and several ossification foci were observed throughout the defect. PLLA/BMP-2 scaffolds also stimulated significantly faster bone regeneration during the first eight weeks compared to bovine spongiosa. However, no significant differences between these two scaffolds could be observed after twelve weeks. Expression of osteogenic marker proteins in PLLA/BMP-2 scaffolds continuously increased throughout the observation period. After twelve weeks osteocalcin, BMP-2 and Smad5 were all significantly higher in the PLLA/BMP-2 group than in all other groups.

Conclusion

Electrospun PLLA nanofibers facilitate colonization of bone defects, while their use in combination with BMP-2 also increases bone regeneration in vivo and thus combines osteoconductivity of the scaffold with the ability to maintain an adequate osteogenic stimulus.     

Experimental bone defect healing with xenogenic demineralized bone matrix and bovine fetal growth plate as a new xenograft: radiological, histopathological and biomechanical evaluation

The following study was designed to evaluate xenogenic bovine demineralized bone matrix (DBM) and new xenograft (Bovine fetal growth plate) effects on bone healing process. Twenty male White New Zealand rabbits were used in this study. In group I (n = 10) the defect was filled by xenogenic DBM and in group II (n = 10) the defect was filled by a segment of bovine fetal growth plate and was fixed by cercelage wire. Radiological, histopathological, and biomechanical evaluations were performed blindly and results scored and analyzed statistically. Statistical tests did not support significant differences between two groups radiographically (P > 0.05). There was a significant difference for union at the 28nd postoperative radiologically (P < 0.05). Xenograft was superior to DBM group at the 28th postoperative day for radiological union (P < 0.03). Histopathological and biomechanical evaluation revealed no significant differences between two groups. In conclusion, the results of this study indicate that satisfactory healing occurred in rabbit radius defect filled with xenogenic bovine DBM and xenogenic bovine fetal growth plate. Complications were not identified and healing was faster in two grafting groups.     

Long-term delivery enhances in vivo osteogenic efficacy of bone morphogenetic protein-2 compared to short-term delivery

In this study, heparin-conjugated poly(l-lactide-co-glycolide) (PLGA) nanospheres (HCPNs) suspended in fibrin gel (group 1) were developed for a long-term delivery of BMP-2, and then used to address the hypothesis that a long-term delivery of BMP-2 would enhance ectopic bone formation compared to a short-term delivery at an equivalent dose. Fibrin gel containing normal PLGA nanospheres (group 2) was used for short-term delivery of BMP-2. The in vitro release of BMP-2 from group 1 was sustained for 4 weeks with no initial burst release. In contrast, 83% of BMP-2 loaded in group 2 was released only for the first 3 days. BMP-2 released from group 1 stimulated an increase in alkaline phosphatase (ALP) activity of osteoblasts for 9 days in vitro. In contrast, BMP-2 released from group 2 induced a transient increase in ALP activity for the first 5 days and a decrease thereafter. Importantly, group 1 induced bone formation to a much greater extent than did group 2, with 2.0-fold greater bone formation area and 3.5-fold greater calcium content, upon implantation into rat hind limb muscle. These results show that long-term delivery of BMP-2 enhances in vivo osteogenic efficacy of the protein compared to short-term delivery at an equivalent dose.     

Osteogenic inhibition by rat periodontal ligament cells: modulation of bone morphogenic protein-7 activity in vivo

Periodontal ligament width is precisely maintained throughout the lifetime of adult mammals but the biological mechanisms that inhibit ingrowth of bone into this soft connective tissue are unknown. As bone morphogenic proteins strongly stimulate osteogenesis and can induce ectopic bone formation in vivo, we tested the hypothesis that topical application of this powerful osteogenic agent will overwhelm the osteogenic inhibitory mechanisms of periodontal ligament cells and induce ankylosis. Wounds through the alveolar bone and periodontal ligament were created in 45 male Wistar rats. Defects were filled with either a collagen implant or collagen plus bone morphogenic protein (BMP-7), or were left unfilled (controls). Three animals per time period were killed on days 2, 5, 10, 21 and 60 after surgery for each wound type. Cellular proliferation and clonal growth in periodontal tissues were assessed by ³H-thymidine labeling 1 h before death, followed by radioautography. Cellular differentiation of soft and mineralizing connective tissue cell populations was determined by immunohistochemical staining of α-smooth muscle actin, osteopontin and bone sialoprotein. In regenerating periodontium, BMP-7 induced abundant bone formation by 21 days (2.5-fold greater than controls or collagen implant only; P<0.001), but by day 60 the volume of the newly formed bone had returned to baseline levels and was similar for all groups. Independent of the type of treatment, periodontal ligament width was unchanged throughout the experimental period (P>0.05). Animals treated with BMP-7 implants showed greatly increased cellular proliferation in the periodontal ligament adjacent to the wound site and in the regenerating alveolar bone at days 5 and 10 after wounding compared to the other treatment groups (P<0.005). Animals in the BMP-7 group exhibited similar spatial and temporal staining patterns for α-smooth muscle actin, osteopontin and bone sialoprotein as controls. Collectively, these data show that BMP-7 promoted the proliferation of precursor cells in the periodontal ligament but did not induce osteogenic differentiation in this compartment. Consequently a powerful osteogenic stimulus like BMP-7 cannot significantly perturb the mechanisms that regulate periodontal ligament width and maintain periodontal homeostasis. Received: 2 March 1998 / Accepted: 16 June 1998     

A statistical model to allow the phasing out of the animal testing of demineralised bone matrix products

Demineralised bone matrix (DBM) products are complex mixtures of proteins known to influence bone growth, turnover, and repair. They are used extensively in orthopaedic surgery, and are bioassayed in vivo prior to being used in clinical applications. Many factors contribute to the osteogenic potency of DBM, but the relative contributions of these factors, as well as the possibility of interactive effects, are not completely defined. The "gold standard" measure of the therapeutic value of DBM, the in vivo assay for ectopic bone formation, is costly, time-consuming, and involves the use of numerous animal subjects. We have measured the levels of five growth factors released by the collagenase digestion of DBM, and statistically related these levels with osteogenic potency as determined by a standard in vivo model, in order to determine which value or combination of values of growth factors best predict osteogenic activity. We conclude that the level of BMP-2 is the best single predictor of osteogenic potency, and that adding the values of other growth factors only minimally increases the predictive power of the BMP-2 measurement. A small, but significant, interactive effect between BMP-2 and BMP-7 was demonstrated. We present a statistical model based on growth factor (e.g. BMP-2) analysis that best predicts the in vivo assay score for DBM. This model allows the investigator to predict which lots of DBM are likely to exhibit in vivo bioactivity and which are not, thus reducing the need to conduct in vivo testing of insufficiently active lots of DBM. This model uses cut-point analysis to allow the user to assign an estimate of acceptable uncertainty with respect to the "gold standard" test. This procedure will significantly reduce the number of animal subjects used to test DBM products.     

Engineered myocardial tissues constructed <Emphasis Type="Italic">in vivo</Emphasis> using cardiomyocyte-like cells derived from bone marrow mesenchymal stem cells in rats

Background  

To explore the feasibility of constructing engineered myocardial tissues (EMTs) in vivo, using polylactic acid -co-glycolic acid (PLGA) for scaffold and cardiomyocyte-like cells derived from bone marrow mesenchymal stem cells (BMMSCs) for seeded cells.     

Bone formation in ectopic and osteogenic tissue induced by a novel BMP-2-related peptide combined with rat tail collagen

Bone morphogenetic proteins (BMPs) play an important role in regulating osteoblast differentiation and subsequent bone formation, mainly evidenced by the induced osteogenic ability of BMP-2 from BMPs. However, BMP-2 alone does not induce the expected efficacy due to its short retention in vivo. In this study, a novel BMP-2-related peptide (designated P24) derived from the “knuckle epitope” of BMP-2 was coupled covalently to type I collagen derived from rat tail and observed under scanning electron microscopy (SEM) in low vacuum mode. The BMP-2-related peptide/collagen composite was implanted in vivo into the pocket of the quadriceps musculature of Sprague-Dawley (SD) rats and then harvested 3 or 6 weeks after surgery. It was found that lyophilized collagen retained a porous network structure with an average inner-diameter of 90 ∼ 160 μm. Based on radiographic evaluation and histological examination, BMP-2-related peptide/collagen induced significant ectopic bone formation compared to that of rat tail collagen alone as a control. Our results indicate collagen served as a good carrier for newly synthesized BMP-2-related peptide and that the BMP-2-related peptide/collagen composite was an effective substitute in bone tissue engineering.     

Heart specific expression of mouse BMP-10 a novel member of the TGF-β superfamily

Here we report the cloning and expression of murine BMP-10, a novel member of the TGF-β superfamily. In the mouse embryo, BMP-10 expression begins at 9.0 d.p.c. and is restricted to the developing heart. Initially, BMP-10 expression localizes to the trabeculated part of the common ventricular chamber and to the bulbus cordis region. After 12.5 d.p.c., additional BMP-10 expression is seen in the atrial wall. The data presented here suggest that BMP-10 plays an important role in trabeculation of the embryonic heart.     

Effects of Hydrogen Peroxide Cleaning Procedures on Bone Graft Osteoinductivity and Mechanical Properties

Bone allografts are frequently used during orthopaedic trauma cases or other reconstructive procedures. Most allografts are processed and cleaned before use. Our goals were to determine if an improved cleaning procedure compromises the strength or osteoinductivity of a graft. We compared our improved cleaning procedure to our standard cleaning procedure on cortical bone allograft. The cleaning procedures are generally composed of a series of chemical steps with nonionic detergents, hydrogen peroxide, and alcohol under time and temperature control, subjected to ultrasonic agitation. We tested the compressive strength, impact strength, and shear strength following the standard and improved cleaning procedures. Osteoinductivity was tested in 4 groups, using the improved cleaning procedure with four different hydrogen peroxide cleaning times: 0, 1, 3, and 5 h. Osteoinductivity was evaluated in vivo, using a 28-day implant in the hamstring muscle of an athymic, nude mouse. Results demonstrated that osteoinductivity is maintained with cleaning in hydrogen peroxide for up to 1 h, and that compressive strength, impact strength, and shear strength were all unaffected by the improved cleaning procedure. The improved cleaning procedure therefore did not compromise the strength or osteoinductivity of cortical bone allografts in comparison to the standard procedure.     

Osteoclasts in bone modeling, as revealed by in vivo imaging, are essential for organogenesis in fish

Bone modeling is the central system controlling the formation of bone including bone growth and shape in early development, in which bone is continuously resorbed by osteoclasts and formed by osteoblasts. However, this system has not been well documented, because it is difficult to trace osteoclasts and osteoblasts in vivo during development. Here we showed the important role of osteoclasts in organogenesis by establishing osteoclast-specific transgenic medaka lines and by using a zebrafish osteoclast-deficient line. Using in vivo imaging of osteoclasts in the transgenic medaka carrying an enhanced GFP (EGFP) or DsRed reporter gene driven by the medaka TRAP (Tartrate-Resistant Acid Phosphatase) or Cathepsin K promoter, respectively, we examined the maturation and migration of osteoclasts. Our results showed that mononuclear or multinucleated osteoclasts in the vertebral body were specifically localized at the inside of the neural and hemal arches, but not at the vertebral centrum. Furthermore, transmission electron microscopic (TEM) analyses revealed that osteoclasts were flat-shaped multinucleated cells, suggesting that osteoclasts initially differentiate from TRAP-positive mononuclear cells residing around bone. The zebrafish panther mutant lacks a functional c-fms (receptor for macrophage colony-stimulating factor) gene crucial for osteoclast proliferation and differentiation and thus has a low number of osteoclasts. Analysis of this mutant revealed deformities in both its neural and hemal arches, which resulted in abnormal development of the neural tube and blood vessels located inside these arches. Our results provide the first demonstration that bone resorption during bone modeling is essential for proper development of neural and vascular systems associated with fish vertebrae.     

Factors affecting entomopathogenic nematode infection ofPlutella xylostella on a leaf surface

We investigated the ability of entomopathogenic nematodes to infect diamondback moth (DBM),Plutella xylostella (L.) (Lepidoptera: Plutellidae) on a leaf surface. In a leaf disk assay, mortality of late stage DBM larvae ranged from <7% caused bySteinernema kushidai Mamiya to >95% caused byS. carpocapsae (Weiser) All strain. LC₅₀ values forS. carpocapsae, S. riobravis Cabanillas, Poinar & Raulston, andHeterorhabditis bacteriophora Poinar NC1 strain were 14.6, 15.4, and 65.4 nematodes/larva, respectively.S. carpocapsae, S. riobravis, andH. bacteriophora caused 29%, 33%, and 14% mortality of DBM pupae, respectively. DBM mortality caused byS. carpocapsae on radish declined at low (<76%) to moderate (76–90%) RH, because nematode survival and infectivity declined at low (<76%) to moderate (76–90%) RH. However, DBM mortality caused byS. riobravis did not decline with RH.S. riobravis survival declined with RH, but infectivity did not. Overall, nematode survival and infectivity to DBM larvae were lower forS. riobravis than forS. carpocapsae. In addition, DBM mortality was higher on radish plants (pubescent leaves) than on cabbage plants (glaborous leaves).     

Blood cell induction in Xenopus animal cap explants: Effects of fibroblast growth factor, bone morphogenetic proteins, and activin

 Cultures of Xenopus blastula animal caps were used to explore the haematopoietic effects of three candidate inducers of mesoderm: basic fibroblast growth factor (bFGF), bone morphogenetic proteins (BMPs) and activin A. In response to either bFGF or activin A, explants expanded into egg-shaped structures, and beneath an outer layer of epidermis, a ventral mesodermal lining surrounded a fluid-filled cavity containing ”blood-like cells”. Immunocytochemistry identified some of these cells as early leukocytes, but erythrocytes were rare. BMP-2 or BMP-4 induced primitive erythrocytes as well as leukocytes, and a high concentration was required for these cells to differentiate in only a small proportion of explants. BMP-2 but not BMP-4 induced ventral mesoderm concomitantly. High concentrations of activin A dorsalized explants, which contained infrequent leukocytes, and an optimal combination of activin A and bFGF caused differentiation of muscle with few blood cells. By contrast, BMP-2 or BMP-4 plus activin A synergistically increased the numbers of both leukocytes and erythrocytes. Explants treated with BMPs plus activin contained a well organized cell mass in which yolk-rich cells mixed with blood cells and pigmented cells did not. BMP-2 plus bFGF also induced numerous leukocytes and fewer erythrocytes, but BMP-4 antagonized the leukopoietic effect of bFGF. The data suggest that the signalling pathways these three factors use to induce leukopoiesis overlap and that erythropoiesis may be activated when inducers are present in combination. Received: 3 August 1998 / Accepted: 7 October 1998     

Sequential transformation to pyramid two Bt genes in vegetable Indian mustard (<Emphasis Type="Italic">Brassica juncea</Emphasis> L.) and its potential for control of diamondback moth larvae

Vegetable Indian mustard (Brassica juncea cv. “Green Wave”) plants that control Plutella xylostella (diamondback moth) (DBM) were produced by introduction of one or two Bacillus thuringiensis (Bt) genes. A cry1Ac Bt gene associated with the nptII gene for kanamycin selection or a cry1C Bt gene with the hpt gene for hygromycin selection was introduced individually through Agrobacterium-mediated transformation of seedling explants. A cry1C line was then transformed with the cry1Ac gene to produce pyramided cry1Ac + cry1C plants. Sixteen cry1C, five cry1Ac, and six cry1Ac + cry1C plants were produced. PCR and Southern analyses confirmed the presence of the cry1C, cry1Ac or pyramided cry1Ac + cry1C genes in the Indian mustard genome. ELISA analysis showed that production of Bt proteins varied greatly among individual transgenic plants, ranging from undetectable to over 1,000 ng Bt/mg total soluble protein. The levels of the Bt proteins were correlated with the effectiveness of control of diamondback moth (DBM) larvae. Insect bioassays indicated that both the cry1C and cry1Ac plants were toxic to susceptible DBM. The cry1C plants also controlled Cry1A-resistant DBM while cry1Ac plants controlled Cry1C-resistant DBM, and the pyramided cry1Ac + cry1C plants effectively controlled all three types of DBM. These Bt-transgenic plants could be used either for direct control of DBM and other lepidopteran insect pests or for tests of “dead-end” trap crops as protection of high value non-transgenic crucifer vegetables such as cabbage.     

<Emphasis Type="Italic">BMP-2</Emphasis> gene-fibronectin-apatite composite layer enhances bone formation

Background  

Safe and efficient gene transfer systems are needed for tissue engineering. We have developed an apatite composite layer including the bone morphogenetic protein-2 (BMP-2) gene and fibronectin (FB), and we evaluated its ability to induce bone formation.     

Cilomilast enhances osteoblast differentiation of mesenchymal stem cells and bone formation induced by bone morphogenetic protein 2

A rapid and efficient method to stimulate bone regeneration would be useful in orthopaedic stem cell therapies. Rolipram is an inhibitor of phosphodiesterase 4 (PDE4), which mediates cyclic adenosine monophosphate (cAMP) degradation. Systemic injection of rolipram enhances osteogenesis induced by bone morphogenetic protein 2 (BMP-2) in mice. However, there is little data on the precise mechanism, by which the PDE4 inhibitor regulates osteoblast gene expression. In this study, we investigated the combined ability of BMP-2 and cilomilast, a second-generation PDE4 inhibitor, to enhance the osteoblastic differentiation of mesenchymal stem cells (MSCs). The alkaline phosphatase (ALP) activity of MSCs treated with PDE4 inhibitor (cilomilast or rolipram), BMP-2, and/or H89 was compared with the ALP activity of MSCs differentiated only by osteogenic medium (OM). Moreover, expression of Runx2, osterix, and osteocalcin was quantified using real-time polymerase chain reaction (RT-PCR). It was found that cilomilast enhances the osteoblastic differentiation of MSCs equally well as rolipram in primary cultured MSCs. Moreover, according to the H89 inhibition experiments, Smad pathway was found to be an important signal transduction pathway in mediating the osteogenic effect of BMP-2, and this effect is intensified by an increase in cAMP levels induced by PDE4 inhibitor.     

BMP depletion occurs during prolonged acid demineralization of bone: characterization and implications for graft preparation

Demineralization of allograft bone increases the bioavailability of matrix-associated bone morphogenetic proteins (BMPs), rendering these grafts osteoinductive. While osteoinductivity is related to BMP content, little is known about how the demineralization protocol, in particular, extended demineralization times, affects graft BMP levels. We characterized the BMP-7 content of <710 μm bovine bone powder demineralized under various conditions. Using 1 g of bone per 50 ml of 0.125 N, 0.25 N, or 0.5 N HCl, demineralization was performed at room temperature for 5 min to 24 h. Minimum residual calcium levels were obtained within 90 min and were <1 wt % using the 0.25 N and 0.5 N baths and 17 wt % using the 0.125 N bath. Measured peak BMP-7 levels were also obtained within 90 min and were 161–165 ng g⁻¹ using the 0.25 N and 0.5 N baths and 55.2 ng g⁻¹ using the 0.125 N bath. This compares to 5.1 ng g⁻¹ for undemineralized bone. Further acid bath exposure to 24 h resulted in BMP-7 decline to about 50% of the peak value, which was significant. The BMP-7 half-life was estimated to be 26 h. It is likely that the decline was due to diffusion of BMP-7 from the bone matrix into the acid. These results suggest the importance of not over demineralizing bone grafts and should stimulate further research that can be incorporated into the processing methodology followed by tissue banks.