Membranous Bone Formation Research Articles

The effect of intermittent parathyroid hormone on bone lengthening: current evidence to inform future effective interventions.

Recent studies have demonstrated the positive effects of parathyroid hormone (PTH) on bone healing, and findings support the use of PTH to accelerate bone healing following distraction osteogenesis. The goal of this review was to compile and discuss the mechanisms potentially underlying the effects of PTH on newly formed bone following a bone-lengthening procedure incorporating all relevant evidence in both animal and clinical studies. This review summarized all evidence from in vivo to clinical studies regarding the effects of PTH administration on a bone-lengthening model. In addition, a comprehensive evaluation of what is currently known regarding the potential mechanisms underlying the potential benefits of PTH in bone lengthening was presented. Some controversial findings regarding the optimal dosage and timing of administration of PTH in this model were also discussed. The findings demonstrated that the potential mechanisms associated with the action of PTH on the acceleration of bone regeneration after distraction osteogenesis are involvement in mesenchymal cell proliferation and differentiation, endochondral bone formation, membranous bone formation, and callus remodeling. In the last 20 years, a number of animal and clinical studies have indicated that there is a prospective role for PTH treatment in human bone lengthening as an anabolic agent that accelerates the mineralization and strength of the regenerated bone. Therefore, PTH treatment can be viewed as a potential treatment to increase the amount of new calcified bone and the mechanical strength of the bone in order to shorten the consolidation stage after bone lengthening.

The Impact of the Ca2+-Independent Phospholipase A2β (iPLA2β) on Immune Cells.

The Ca2+-independent phospholipase A2β (iPLA2β) is a member of the PLA2 family that has been proposed to have roles in multiple biological processes including membrane remodeling, cell proliferation, bone formation, male fertility, cell death, and signaling. Such involvement has led to the identification of iPLA2β activation in several diseases such as cancer, cardiovascular abnormalities, glaucoma, periodontitis, neurological disorders, diabetes, and other metabolic disorders. More recently, there has been heightened interest in the role that iPLA2β plays in promoting inflammation. Recognizing the potential contribution of iPLA2β in the development of autoimmune diseases, we review this issue in the context of an iPLA2β link with macrophages and T-cells.

Biofunctionalization with a TGFβ-1 Inhibitor Peptide in the Osseointegration of Synthetic Bone Grafts: An In Vivo Study in Beagle Dogs.

Objectives: The aim of this research was to determine the osseointegration of two presentations of biphasic calcium phosphate (BCP) biomaterial—one untreated and another submitted to biofunctionalization with a TGF-β1 inhibitor peptide, P144, on dental alveolus. Materials and Methods: A synthetic bone graft was used, namely, (i) Maxresorb® (Botiss Klockner) (n = 12), and (ii) Maxresorb® (Botiss Klockner) biofunctionalized with P144 peptide (n = 12). Both bone grafts were implanted in the two hemimandibles of six beagle dogs in the same surgical time, immediately after tooth extraction. Two dogs were sacrificed 2, 4, and 8 weeks post implant insertion, respectively. The samples were submitted to histomorphometrical and histological analyses. For each sample, we quantified the new bone growth and the new bone formed around the biomaterial’s granules. After optical microscopic histological evaluation, selected samples were studied using backscattered scanning electron microscopy (BS-SEM). Results: The biofunctionalization of the biomaterial’s granules maintains a stable membranous bone formation throughout the experiment timeline, benefitting from the constant presence of vascular structures in the alveolar space, in a more active manner that in the control samples. Better results in the experimental groups were proven both by quantitative and qualitative analysis. Conclusions: Synthetic bone graft biofunctionalization results in slightly better quantitative parameters of the implant’s osseointegration. The qualitative histological and ultramicroscopic analysis shows that biofunctionalization may shorten the healing period of dental biomaterials.

The Bone Regeneration Model and Primary Osteoblastic Cell Culture Used in the Analysis of Ccn3 Transgenic and Knockout Mice.

Bone tissue is intrinsically hard and thus, it is more difficult to handle, process, and examine than soft tissues. Here, we describe an experimental model of bone regeneration and several selected protocols useful for investigating mRNA and protein expression in bone. The inhibitory function of CCN3 on membranous bone formation has been confirmed by following the protocols described herein (Fig. 1).

Inter-trabecular bone formation: a specific mechanism for healing of cancellous bone

Background and purpose — Studies of fracture healing have mainly dealt with shaft fractures, both experimentally and clinically. In contrast, most patients have metaphyseal fractures. There is an increasing awareness that metaphyseal fractures heal partly through mechanisms specific to cancellous bone. Several new models for the study of cancellous bone healing have recently been presented. This review summarizes our current knowledge of cancellous fracture healing.Methods — We performed a review of the literature after doing a systematic literature search.Results — Cancellous bone appears to heal mainly via direct, membranous bone formation that occurs freely in the marrow, probably mostly arising from local stem cells. This mechanism appears to be specific for cancellous bone, and could be named inter-trabecular bone formation. This kind of bone formation is spatially restricted and does not extend more than a few mm outside the injured region. Usually no cartilage is seen, although external callus and cartilage formation can be induced in meta­physeal fractures by mechanical instability. Inter-trabecular bone formation seems to be less sensitive to anti-inflammatory treatment than shaft fractures.Interpretation — The unique characteristics of inter-trabecular bone formation in metaphyseal fractures can lead to differences from shaft healing regarding the effects of age, loading, or drug treatment. This casts doubt on generalizations about fracture healing based solely on shaft fracture models.

Tissue segregation restores the induction of bone formation by the mammalian transforming growth factor-β3 in calvarial defects of the non-human primate Papio ursinus

A diffusion molecular hypothesis from the dura and/or the leptomeninges below that would control the induction of calvarial membranous bone formation by the recombinant human transforming growth factor-β3 (hTGF-β3) was investigated. Coral-derived calcium carbonate-based macroporous constructs (25 mm diameter; 3.5/4 mm thickness) with limited hydrothermal conversion to hydroxyapatite (7% HA/CC) were inserted into forty calvarial defects created in 10 adult Chacma baboons Papio ursinus. In 20 defects, an impermeable nylon foil membrane (SupraFOIL®) was inserted between the cut endocranial bone and the underlying dura mater. Twenty of the macroporous constructs were preloaded with hTGF-β3 (125 μg in 1000 μl 20 mM sodium succinate, 4% mannitol pH4.0), 10 of which were implanted into defects segregated by the SupraFOIL® membrane, and 10 into non-segregated defects. Tissues were harvested on day 90, processed for decalcified and undecalcified histology and quantitative real-time polymerase chain reaction (qRT-PCR). Segregated untreated macroporous specimens showed a reduction of bone formation across the macroporous spaces compared to non-segregated constructs. qRT-PCR of segregated untreated specimens showed down regulation of osteogenic protein-1 (OP-1), osteocalcin (OC), bone morphogenetic protein-2 (BMP-2), RUNX-2 and inhibitor of DNA binding-2 and -3 (ID2,ID3) and up regulation of TGF-β3, a molecular signalling pathway inhibiting the induction of membranous bone formation. Non-segregated hTGF-β3/treated constructs also showed non-osteogenic expression profiles when compared to non-segregated untreated specimens. Segregated hTGF-β3/treated 7% HA/CC constructs showed significantly greater induction of bone formation across the macroporous spaces and, compared to non-segregated hTGF-β3/treated constructs, showed up regulation of OP-1, OC, BMP-2, RUNX-2, ID2 and ID3. Similar up-regulated expression profiles were seen for untreated non-segregated constructs. TGF-β signalling via ID genes creates permissive or refractory micro-environments that regulate the induction of calvarial bone formation which is controlled by the exogenous hTGF-β3 upon segregation of the calvarial defects. The dura is the common regulator of the induction of calvarial bone formation modulated by the presence or absence of the SupraFOIL® membrane with or without hTGF-β3.

Osteocrin, a peptide secreted from the heart and other tissues, contributes to cranial osteogenesis and chondrogenesis in zebrafish.

The heart is an endocrine organ, as cardiomyocytes (CMs) secrete natriuretic peptide (NP) hormones. Since the discovery of NPs, no other peptide hormones that affect remote organs have been identified from the heart. We identified osteocrin (Ostn) as an osteogenesis/chondrogenesis regulatory hormone secreted from CMs in zebrafish. ostn mutant larvae exhibit impaired membranous and chondral bone formation. The impaired bones were recovered by CM-specific overexpression of OSTN. We analyzed the parasphenoid (ps) as a representative of membranous bones. In the shortened ps of ostn morphants, nuclear Yap1/Wwtr1-dependent transcription was increased, suggesting that Ostn might induce the nuclear export of Yap1/Wwtr1 in osteoblasts. Although OSTN is proposed to bind to NPR3 (clearance receptor for NPs) to enhance the binding of NPs to NPR1 or NPR2, OSTN enhanced C-type NP (CNP)-dependent nuclear export of YAP1/WWTR1 of cultured mouse osteoblasts stimulated with saturable CNP. OSTN might therefore activate unidentified receptors that augment protein kinase G signaling mediated by a CNP-NPR2 signaling axis. These data demonstrate that Ostn secreted from the heart contributes to bone formation as an endocrine hormone.

Distinct VEGF Functions During Bone Development and Homeostasis

Vascular endothelial growth factor-A (VEGF) is a key regulator of physiological hemangiogenesis during development, postnatal growth, and homeostasis. It is well known that VEGF is required for effective coupling of angiogenesis to endochondral and membranous bone formation during skeletal development. However, less well known are the roles of VEGF in regulating the differentiation and/or functions of skeletal cells such as chondrocytes, osteoblasts, and osteoclasts. In this review, we discuss some of these functions. During early skeletal development, VEGF is important for the survival of chondrocytes in the hypoxic regions of the cartilage models of future bones, the vascularization of developing bones and proliferation and differentiation of osteoblastic cells. Postnatally, osteoblast-derived VEGF is critical for maintaining bone homeostasis by stimulating the differentiation of mesenchymal stem cells to osteoblasts and repressing their differentiation to adipocytes. Recent data indicate that these effects of VEGF on osteogenic/adipogenic stem cell fates are based on an intracellular (intracrine) mechanism. In contrast, osteoblast-derived VEGF is also known to stimulate the differentiation of monocytes to osteoclasts by a paracrine mechanism. Mice with VEGF-deficient osteoblastic lineage cells exhibit age-dependent loss of bone mass and an increase in bone marrow fat. These changes are similar to the changes associated with osteoporosis in humans. Thus, a better understanding of the intracellular mechanisms by which VEGF regulates osteoblastic and adipogenic differentiation may lead to the identification of new targets for therapies to prevent osteoporotic bone loss.

Rabbit maxillary sinus augmentation model with simultaneous implant placement: differential responses to the graft materials

PurposeThis study was performed to establish an experimental rabbit model for single-stage maxillary sinus augmentation with simultaneous implant placement.MethodsTwelve mature New Zealand white rabbits were used for the experiments. The rabbit maxillary sinuses were divided into 3 groups according to sinus augmentation materials: blood clot (BC), autogenous bone (AB), and bovine-derived hydroxyapatite (BHA). Small titanium implants were simultaneously placed in the animals during the sinus augmentation procedure. The rabbits were sacrificed 4 and 8 weeks after surgery and were observed histologically. Histomorphometric analyses using image analysis software were also performed to evaluate the parameters related to bone regeneration and implant-bone integration.ResultsThe BC group showed an evident collapse of the sinus membrane and limited new bone formation around the original sinus floor at 4 and 8 weeks. In the AB group, the sinus membrane was well retained above the implant apex, and new bone formation was significant at both examination periods. The BHA group also showed retention of the elevated sinus membrane above the screw apex and evident new bone formation at both points in time. The total area of the mineral component (TMA) in the area of interest and the bone-to-implant contact did not show any significant differences among all the groups. In the AB group, the TMA had significantly decreased from 4 to 8 weeks.ConclusionsWithin the limits of this study, the rabbit sinus model showed satisfactory results in the comparison of different grafting conditions in single-stage sinus floor elevation with simultaneous implant placement. We found that the rabbit model was useful for maxillary sinus augmentation with simultaneous implant placement.

Heterotopic bone formation ability of BCP combined with rhBMP-2 and bFGF microspheres

Objective To investigate the heterotopic bone formation ability of biphasic ceramics phosphate (BCP) combined with recombinant human bone morphogenetic protein-2 (rhBMP-2) and basic fibroblast growth factor (bFGF) microspheres.Methods The rabbits were divided into group A (rhBMP-2+bFGF/BCP), group B (BCP), group C (bFGF/BCP), and group D (rhBMP-2/BCP). The concentrations of bFGF and rhBMP were 5 μg and 15 μg, respectively. All the samples were 3 mm×8 mm×28 mm in size. The muscle pouches of the rabbits were implanted with the samples. The ectopic bone formation was evaluated in the following aspects: histology, osteogenic area, and blood capillary number at 4th, 8th and 12th week after operation.Results At any specified time point, the value of the heterotopic bone formation was significantly higher in group A than other groups (P< 0.05). The histological analysis showed that group A had more mesenchymal (MES) cells and fewer blood capillaries at 4th week after operation. At 8th week, the composite was degradated and diffusely distributed, and the immature new bone was found. There were low-grade mature woven bones at 12th week, and the membranous bone formation occurred.Conclusion BCP combined with rhBMP-2 and bFGF microspheres has good heterotopic bone formation ability. Key words: Biphasic ceramics phosphate; Recombinant human bone morphogenetic protein-2; Basic fibroblast growth factor; Microsphere; Heterotopic bone formation ability

Osteogenesis Mechanism of Chitosan-Coated Calcium Sulfate Pellets on the Restoration of Segmental Bone Defects

A radial segmental defect model of a rabbit was used to study the restoration effect on defects treated with chitosan-coated pressed calcium sulfate pellets combined with recombinant human bone morphogenetic protein-2 (rhBMP-2), coated pressed calcium sulfate pellets, and uncoated pressed calcium sulfate pellets. Nothing was implanted in the control group. After 4, 8, and 12 weeks, the results indicated that coated pressed calcium sulfate pellets combined with rhBMP-2 and coated pressed calcium sulfate pellets facilitated new bone formation on defected bones and that, particularly, the coated pressed calcium sulfate pellets combined with rhBMP-2 was more effective than the coated pressed calcium sulfate pellet. Histologic and tetracycline fluorimetric findings showed that the osteogenesis mechanism of chitosan-coated pressed calcium sulfate pellets is membrane bone formation, and the pellets showed slightly slower resorption that closely coincides with the growth rate of new bone.

Axial shortening during distraction osteogenesis leads to enhanced bone formation in a rabbit model through the HIF-1α/vascular endothelial growth factor system

Axial micromotion of bone fragments enhances callus formation during fracture repair or limb lengthening. To examine this, we used an axial-shortening model of the tibial callus in rabbits and performed histological analyses. After 10-mm lengthening of the left tibia with an external fixator, we shortened the callus by 2 mm. Radiographs and quantitative evaluation of corrected bone mineral density showed a significant increase in mineralization in the shortened callus (57.3 vs. 36.2%, p = 0.001). Histologically, greater osteoblast proliferation and more vigorous trabecular bone formation were noted in the shortened calluses than in the controls. Around the front of membranous bone formation in the shortened callus, there was a significant decrease in mean percentage area of vascular lumens (1.8 vs. 4.5%, p = 0.009), which seemed attributable to compressive force, and a significantly increased production of vascular endothelial growth factor (VEGF; 422.5 vs. 142.7 pg/mg protein, p = 0.007) and its receptors. There were also increased numbers of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts and proliferating cell nuclear antigen (PCNA)-positive cells. A marked increase of hypoxia inducible factor-1alpha (HIF-1alpha) expression in osteoblasts was also observed in this area. Thus, enhancement of membranous bone formation by static compression or axial dynamization may be at least partly attributable to HIF-1alpha-mediated VEGF induction following the local hypoxia caused by collapse of vascular lumens.

FGF and FGFR signaling in chondrodysplasias and craniosynostosis

The first experimental mouse model for FGF2 in bone dysplasia was made serendipitously by overexpression of FGF from a constitutive promoter. The results were not widely accepted, rightfully drew skepticism, and were difficult to publish; because of over 2,000 studies published on FGF-2 at the time (1993), only a few reported a role of FGF-2 in bone growth and differentiation. However, mapping of human dwarfisms to mutations of the FGFRs shortly, thereafter, made the case that bone growth and remodeling was a major physiological function for FGF. Subsequent production of numerous transgenic and targeted null mice for several genes in the bone growth and remodeling pathways have marvelously elucidated the role of FGFs and their interactions with other genes. Indeed, studies of the FGF pathway present one of the best success stories for use of experimental genetics in functionally parsing morphogenetic regulatory pathways. What remains largely unresolved is the pleiotropic nature of FGF-2. How does it accelerate growth in one cell then stimulate apoptosis or retard growth for another cell in the same type of tissue? Some of the answers may come through distinguishing the FGF-2 protein isoforms, made from alternative translation start sites, these appear to have substantially different functions. Although we have made substantial progress, there is still much to be learned regarding FGF-2 as a most complex, enigmatic protein. Studies of genetic models in mice and human FGFR mutations have provided strong evidence that FGFRs are important modulators of osteoblast function during membranous bone formation. However, there is some controversy regarding the effects of FGFR signaling in human and murine genetic models. Although significant progress has been made in our understanding of FGFR signaling, several questions remain concerning the signaling pathways involved in osteoblast regulation by activated FGFR. Additionally, little is known about the specific role of FGFR target genes involved in cranial bone formation. These issues need to be addressed in future in in vitro and in vivo approaches to better understand the molecular mechanisms of action of FGFR signaling in osteoblasts that result in anabolic effects in bone formation.

Apoptosis in Membranous Bone Formation: Role of Fibroblast Growth Factor and Bone Morphogenetic Protein Signaling

Membranous ossification occurs by the condensation of mesenchymal cells followed by their progressive differentiation into osteoblasts that form a mineralized matrix in ossification centers. The balance between proliferating and differentiated osteogenic cells at the suture areas between calvarial bones is essential for the control of suture maintenance and membranous bone formation. The mechanisms of regulation of na apoptosis in suture areas begin to be understood. Fibroblast growth factor (FGF) and bone morphogenetic protein (BMP) are important regulators of mesenchymal, preosteoblast, and osteoblast apoptosis in suture areas. Perturbations in FGF or BMP signaling lead to alter the number of apoptotic osteogenic cells, resulting in premature or delayed suture closure. Recent data indicate that FGF signaling downregulates preosteoblast apoptosis, thereby preventing premature fusion of adjacent mineralizing extremities. In contrast, continuous FGF signaling or constitutive FGF receptor activation, as well as BMP signaling, upregulate osteoblast apoptosis. Additionally, multiple signaling mechanisms, including PI3K and PKC, appear to be involved in the control of calvarial osteoblast apoptosis by FGF and BMP. These mechanisms allow a fine control of the number of functional bone-forming cells and, thereby, the normal progression of membranous bone formation.

Apoptosis in Membranous Bone Formation: Role of Fibroblast Growth Factor and Bone Morphogenetic Protein Signaling

Apoptosis in Membranous Bone Formation: Role of Fibroblast Growth Factor and Bone Morphogenetic Protein Signaling

Ortho- and heterotopic bone grafts in bifocal transport osteogenesis for craniofacial reconstruction—an experimental study in sheep

Bifocal transport osteogenesis (BTO) is a promising technique for the reconstruction of extended osseous craniofacial defects. Aim of this study was to determine the potential of this technique related to various donor sites of the transport segment. In 10 adult sheep critical size defects of the calvaria were treated by gradual movement of a transport segment consisting either of autogenous regional free calvarial bone grafts ( n=5) or autogenous illiac free bone grafts ( n=5). Latency period was 5 days; the rate of distraction was 1 mm per day and extended approximately 40 days. The consolidation period was 28 days. After harvesting, specimens were investigated by conventional radiography, CT-scans, histologically and by fluorescence. In both groups transport osteogenesis resulted in a complete closure of the critical size defect. Membranous bone formation and remodeling occurred during the entire period of distraction and consolidation. The volumes and thickness of newly formed bone at the defect site were increased significantly when calvarial bone grafts were used ( P<0.05). Iliac bone grafts became progrediently smaller during distraction, while the volume of calvarial grafts remained relatively constant ( P<0.05). In conclusion, transport segments consisting of calvarial and iliac bone resulted in a reliable closure of craniofacial critical size defects in adult organisms; the application of calvarial bone grafts resulted in an increased extend of bone formation.

Mechanism of bone formation with gene transfer of the cDNA encoding for the intracellular protein LMP-1.

LIM mineralization protein-1 (LMP-1), an intracellular protein, is thought to induce secretion of soluble factors that convey its osteoinductive activity. Although evidence suggests that LMP-1 may be a critical regulator of osteoblast differentiation in vitro and in vivo, little is known about its mechanism of action. The purpose of the present study was to identify candidates for the induced secreted factors and to describe the time sequence of histological changes during bone formation induced by LMP-1. Human lung carcinoma (A549) cells were used to determine if LMP-1 overexpression would induce expression of bone morphogenetic proteins (BMPs) in vitro. Cultured A549 cells were infected with recombinant replication-deficient human type-5 adenovirus containing the LMP-1 or LacZ cDNA. Cells were subjected to immunohistochemical analysis after forty-eight hours. Finally, sixteen athymic rats received subcutaneous implants consisting of collagen disks loaded with human buffy-coat cells that were infected with one of the above two viruses. Rats were killed at intervals, and explants were studied with histological and immunohistochemical analyses. In vitro experiments with A549 cells showed that AdLMP-1-infected cells express elevated levels of BMP-2, BMP-4, BMP-6, BMP-7, and TGF-beta1 (transforming growth factor-beta 1) protein. Human buffy-coat cells infected with AdLMP-1 also demonstrated increased levels of BMP-4 and BMP-7 protein seventy-two hours after ectopic implantation in athymic rats, confirming the in vitro hypothesis. The osteoinductive properties of LMP-1 involve synthesis of several BMPs and the recruitment of host cells that differentiate and participate in direct membranous bone formation.

Mechanism of bone formation by LIM mineralization protein–1 gene therapy

Purpose of study: LIM mineralization protein-1 (LMP-1), an osteoinductive protein identified in 1998, is thought to induce secretion of soluble factors that convey its osteoinductive activity and capability of inducing spine fusion. LMP-1 has been successfully delivered using several ex vivo gene therapy techniques and has proven its ability to consistently induce spine fusion in experimental models. The purpose was to determine the mechanism of bone formation of LMP-1.Methods used: In Phase I, conditioned medium from rat osteoblast cultures overexpressing the LMP-1 cDNA was placed on nondifferentiating cultures. The overexpression cultures were cotreated with antisense oligonucleotides complimentary to bone morphogenic protein (BMP)-4, BMP-6, BMP-7 or a nonsense sequence to see if any of those BMPs were candidates for the LMP-1–induced differentiation factor. In Phase 2, A549 cells were used to test for the induction of candidate BMPs in vitro. The cells were infected in chamber slides for 2 days with adenovirus (MOI=10) containing the cDNA for LMP-1 (AdLMP-1) or B-galactosidase (AdBgal). Untreated cells were used as an additional negative control. Cells were analyzed by immunohistochemical methods for BMP family members. In Phase 3, 16 athymic rats each received four subcutaneous implants on the chest. Human buffy coat cells from peripheral blood were used to deliver the LMP-1 cDNA. One million cells were infected for 10 minutes (MOI = 4.0) with either AdLMP-1 or AdBgal (control) and then placed on a collagen disc and implanted. The animals were sacrificed at intervals, and explants were analyzed by histology and immunohistochemistry.of findings: In Phase 1 the antisense experiments suggested that BMP-4 and BMP-7, but not BMP-6, were required for LMP-1 activity. In Phase 2, the A549 cells treated by AdLMP-1 had strong intracellular reaction for BMP-2, BMP-4 and BMP-7. Some cells also stained slightly positive with the anti–BMP-6 antibody. There was no specific staining for any of these BMPs in the cells treated with AdBgal or the untreated control cells. In Phase 3 human leukocytes were implanted on collagen discs in rats and by Day 3 an increased number of cells were seen at the periphery of the AdLMP-1–treated implants. By Day 5, the number of cells surviving in the center of the implant was diminished, especially in the controls. Extracellular matrix could be seen deposited near the cells in the periphery by day 7 in the Ad-LMP implants. By Day 10, osteoblast-like cells were observed in the voids between the collagen fibers. Osteoid and some mineralized bone were consistently seen by Day 14 growing inward from the edge of the implant. No cartilage phase was observed. There was strong BMP-4 and BMP-7 staining on Day 3 and Day 5 in AdLMP-1 implants within cells, confirming the in vitro observations.Relationship between findings and existing knowledge: Little is known about the mechanism of action of LMP-1 or its relationship to BMPs. The osteoinductive activity of LMP-1 appears to involve synthesis of several BMPs, including BMP-4 and BMP-7. In addition, BMP-2, BMP-6 and TGF-b were also be induced by LMP-1.Overall significance of findings: The LMP-1 cDNA can be transferred into peripheral blood leukocytes in 10 minutes without special equipment. LMP-1 is able to induce the complete membranous bone formation cascade in vivo. Future studies will determine if there are advantages to induce local synthesis of multiple BMP family members, as with LMP-1, as an alternative to implanting larger doses of a single recombinant BMP.Disclosures: Device or drug: LMP-1. Status: not approved.Conflict of interest: Scott D. Boden, grant research support and consultant, Medtronic Sofamor Danek; Scott D. Boden, University licensed, LMP-1 to Medtronic Sofamor Danek.

Three-dimensional histomorphometric analysis of distraction osteogenesis using an implanted device for mandibular lengthening in sheep.

The aim of this study was to lengthen the sheep mandible with a fully buried device and to quantitatively analyze the tissue regenerate in the distraction gap by means of two-dimensional and three-dimensional histomorphometry. A custom-made device for continuous distraction was used in five adult sheep and fixed with three bicortical screws on either side of an osteotomy, anterior to the premolar region of the mandible. A cable-connected power and control unit was implanted in the neck region. After a 5-day latency period, distraction was activated every 2 hours and advanced at a rate of 1.01 mm per day. The distraction period was planned for 14 days, but because of stability problems and cable breakage, the actual distraction period ranged from 2 to 17 days, resulting in gap distances from 1.7 to 17.1 mm (mean, 0.95 mm/day). Osteogenesis was followed by radiographic imaging, and after a 6-week consolidation period, the harvested mandibles were serially sectioned for histologic and two-dimensional histomorphometric analysis, with three-dimensional reconstruction. Histologic examination of the specimens demonstrated predominantly membranous bone formation with remodeling bridging the distraction gap mainly in the periosteal region of the lingual side. In addition, cartilaginous areas and chondral bone formation were observed where the bridging appeared incomplete. Because of device fixation on the buccal side of the mandible, the preservation of the lingual periosteum seemed to play the major role for sufficient bone repair in the distraction gap. Cartilage within the distraction gap suggests fixation instability in this animal model.

Characterization of Midface Maxillary Membranous Bone Formation during Distraction Osteogenesis

The purpose of the study was to follow the early events in bone formation and neovascularization during maxillary distraction and after the consolidation period and to define the characterization of the new bone in the distracted area. Maxillary osteotomy was performed in seven sheep. In five animals, an external distraction device was used for maxillary lengthening of 20 mm at a rate of 1 mm/day for 20 days. Another two animals served as controls without distraction. Sequential biopsies were performed. The methods used for analysis were histologic, immunohistochemical, and ultrastructural by transmission electron microscopy. During the 5 days of latency, a fibrin clot was formed that after 5 days of distraction was replaced by granulation tissue, proliferating mesenchyme-like cells, and capillaries. After 10 days of distraction, the regenerated tissue could be divided into three main zones and two transitional areas: a central zone occupied by many polygonal mesenchyme-like cells and spindle-shaped cells that proliferated intensively; two paracentral zones on both sides of the central zone in which many cells showed morphologic signs of apoptosis leading to a decreased number of fibroblast-like cells embedded in wavy collagen fibers; a transitional area from the central to the paracentral zone in which concentric cellular colonies were believed to represent a novel form of vasculogenesis; distal-proximal zones, located on both sides of the paracentral zones and in continuation with the old bone, showed delicate new woven bone trabeculae that grew continuously in the direction of lengthening and gradually became mineralized; and a transitional area from the paracentral to the distal-proximal zones in which there was recruitment of preosteoblasts from the distracted tissue to the trabecular tips. These further differentiated into osteoblasts that contributed to the trabecular growth. The histologic feature pattern was similar after 15 and 20 days of continuous distraction. At the end of lengthening, after 20 days, delicate longitudinally oriented trabeculae continued to grow by recruiting preosteogenic cells from the central distracted tissue, became mineralized, and were rimmed by osteoblasts. After 6 weeks of retention, the trabeculae thickened and consisted of a mixture of lamellar and woven bone. In conclusion, the distraction force creates a pool of undifferentiated mesenchyme-like cells with osteogenic potential and triggers capillary formation, a clear zonation can be observed during active lengthening, and new bone trabeculae begin to form between 5 and 10 days after distraction, soon become aligned with osteoblasts, and continue to grow as long as distraction force is applied. This characterization may help in any exogenous involvement with growth factors to improve bone quality.