Search

Bone regeneration procedures in clinics and bone tissue engineering stand on three pillars: osteoconduction, osteoinduction, and stem cells. In the last two decades, the focus in this field has been on osteoinduction, which is realized by the use of bone morphogenetic proteins and the application of mesenchymal stem cells to treat bone defects. However, osteoconduction was reduced to a surface phenomenon because the supposedly ideal pore size of osteoconductive scaffolds was identified in the 1990s as 0.3–0.5?mm in diameter, forcing bone formation to occur predominantly on the surface. Meanwhile, additive manufacturing has evolved as a new tool to realize designed microarchitectures in bone substitutes, thereby enabling us to study osteoconduction as a true three-dimensional phenomenon. Moreover, by additive manufacturing, wide-open porous scaffolds can be produced in which bone formation occurs distant to the surface at a superior bony defect-bridging rate enabled by highly osteoconductive pores 1.2?mm in diameter. This review provides a historical overview and an updated definition of osteoconduction and related terms. In addition, it shows how additive manufacturing can be instrumental in studying and optimizing osteoconduction of bone substitutes, and provides novel optimized features and boundaries of osteoconductive microarchitectures.Impact StatementThis review updates the definition of osteoconduction and draws clear lines to discriminate between osteoconduction, osseointegration, and osteoinduction. Moreover, additively manufactured libraries of scaffolds revealed that: osteoconduction is more a three-dimensional than a surface phenomenon;microarchitecture dictates defect bridging, which is the measure for osteoconduction;pore diameter or the diagonal of lattice microarchitectures of osteoconductive bone substitutes should be ~1.2?mm.

Additive manufacturing of bone tissue engineering scaffolds will become a key element for personalized bone tissue engineering in the near future. Several additive manufacturing processes are based on extrusion where the deposition of the filament will result in a three-dimensional lattice structure. Recently, we studied diverse lattice structures for bone tissue engineering realized by laser sintering of titanium. In this work, we used lithography-based ceramic manufacturing of lattice structures to produce scaffolds from tricalcium phosphates (TCP) and compared them in vivoto congruent titanium scaffolds manufactured with the identical computer-aided design data to look for material-based differences in bony healing. The results show that, during a 4-week period in a noncritical-size defect in a rabbit calvarium, both scaffolds with the identical microarchitecture performed equally well in terms of bony regeneration and bony bridging of the defect. A significant increase in both parameters could only be achieved when the TCP-based scaffolds were doped with bone morphogenetic protein-2. In a critical-size defect in the calvarial bone of rabbits, however, the titanium scaffold performed significantly better than the TCP-based scaffold, most likely due to its higher mechanical stability. We conclude that titanium and TCP-based scaffolds of the same microarchitecture perform equally well in terms of bone regeneration, provided the microarchitecture meets the mechanical demand at the site of implantation.

Conventional root canal treatment in immature permanent teeth can lead to early tooth loss in children because root formation is discontinued. We investigated whether the stem cell factor (SCF) could facilitate cell homing in the pulpless immature root canal and promote regeneration of a functional pulp. In vitro, human mesenchymal stem cells (hMSCs) were exposed to SCF at various concentrations for assessing cell migration, proliferation, and differentiation toward odonto/osteoblasts by 3D-chemotaxis slides, WST-1 assay, and alkaline phosphatase activity, respectively. Fibrin gels were used to deliver 15 μg/mL SCF for in vivoexperiments. The release kinetic of SCF was assessed in vitro. Two corresponding human immature premolars, with or without SCF, were placed at rat calvariae for 6 and 12 weeks. All tooth specimens were either analyzed histologically and the percentage of tissue ingrowth determined or the cells were extracted from the pulp space, and the mRNA level of DMP1, DSPP, Col1, NGF, and VEGF were assessed by quantitative polymerase chain reaction. In the presence of SCF, we saw an increase in hMSCs directional migration, proliferation, and odonto/osteogenic differentiation. SCF also increased the extent of tissue ingrowth at 6 weeks but not at 12 weeks. However, at this time point, the formed tissue appeared more mature in samples with SCF. In terms of gene transcription, DMP1, Col1, and VEGF were the significantly upregulated genes, while DSPP and NGF were not affected. Our results suggest that SCF can accelerate cell homing and the maturation of the pulp–dentin complex in human immature teeth.

The treatment of large bone defects still poses a major challenge in orthopaedic and cranio-maxillofacial surgery. One possible solution could be the development of personalized porous titanium-based implants that are designed to meet all mechanical needs with a minimum amount of titanium and maximum osteopromotive properties so that it could be combined with growth factor-loaded hydrogels or cell constructs to realize advanced bone tissue engineering strategies. Such implants could prove useful for mandibular reconstruction, spinal fusion, the treatment of extended long bone defects, or to fill in gaps created on autograft harvesting. The aim of this study was to determine the mechanical properties and potential of bone formation of light weight implants generated by selective laser melting (SLM). We mainly focused on osteoconduction, as this is a key feature in bone healing and could serve as a back-up for osteoinduction and cell transplantation strategies. To that end, defined implants were produced by SLM, and their surfaces were left untreated, sandblasted, or sandblasted/acid etched. In vivobone formation with the different implants was tested throughout calvarial defects in rabbits and compared with untreated defects. Analysis by micro computed tomography (μCT) and histomorphometry revealed that all generatively produced porous Ti structures were well osseointegrated into the surrounding bone. The histomorphometric analysis revealed that bone formation was significantly increased in all implant-treated groups compared with untreated defects and significantly increased in sand blasted implants compared with untreated ones. Bone bridging was significantly increased in sand blasted acid-etched scaffolds. Therefore, scaffolds manufactured by SLM should be surface treated. Bone augmentation beyond the original bone margins was only seen in implant-treated defects, indicating an osteoconductive potential of the implants that could be utilized clinically for bone augmentation purposes. Therefore, designed porous, lightweight structures have potential for bone regeneration and augmentation purposes, especially when complex and patient-specific geometries are essential.

In medicine, N-methyl pyrrolidone (NMP) has a long track record as a constituent in medical devices approved by the Food and Drug Administration and thus can be considered as a safe and biologically inactive small chemical. In the present study, we report on the newly discovered pharmaceutical property of NMP in enhancing bone regeneration in a rabbit calvarial defect model in vivo. At the cellular level, the pharmaceutical effect of NMP was confirmed, in particular, in combination with bone morphogenetic protein (BMP)-2, because NMP increased early and late markers for maturation of preosteoblasts and human bone marrow–derived stem cells in vitro.When we used the multipotent cell line C2C12 without autologous BMP expression, NMP alone had no effect on alkaline phosphatase activity, a marker for osteogenic transdifferentiation. Nevertheless, in combination with low BMP-2 doses, alkaline phosphatase activity was more than eight times as great. Thus, the pharmaceutical NMP mode of action is that of an enhancer of BMP activity. The dependency of the effects of NMP on BMP was confirmed in preosteoblasts because noggin, an extracellular BMP inhibitor, suppressed NMP-induced increases in early markers for osteoblast maturation in vitro. At the molecular level, NMP was shown to have no effect on the binding of BMP-2 to the ectodomain of the high-affinity BMP receptor IA. However, NMP further increased the phosphorylation of p38 and Smad1,5,8 induced by BMP-2. Thus, the small chemical NMP enhances BMP activity by increasing the kinase activity of the BMP receptor complex for Smad1,5,8 and p38 and could be employed as a potent drug for bone tissue regeneration and engineering.

Heterotopic ossification is a frequent complication in patients who have suffered head and neck traumas or have undergone total hip replacement. In this report, stable folding variants of the natural occurring osteoinductive BMPs were shown to act as inhibitors for heterotopic ossification. The most effective BMP folding variant construct performed even better than the natural occurring BMP antagonist Noggin because it also inhibited calcium deposition of pre‐osteoblastic cells.Introduction:Signal transduction through receptor and ligand binding depends on the proper folding of all partners, especially when it involves the formation of a heterotetramer. In the case, the receptor binding of the ligand can be uncoupled from signal transduction, and folding variants of a ligand can be developed into antagonists of the natural bioactivity of the ligand. Here we present a deletion mutant of a bone morphogenetic protein (BMP) folding variant capable of inhibiting the bone‐inducing action of natural occurring BMPs.Materials and Methods:Deletion mutants and site‐directed mutants of BMP folding variants were generated and tested for their ability to reduce alkaline phosphatase activity and mineralization in a pre‐osteoblastic cell line. In vivo activity of the optimized folding variant was determined in a heterotopic ossification model in rodents and in two Xenopus laevismodel systems. Biosensor interaction analysis was used to determine the affinity of the optimized BMP folding variant to the extracellular domain of BMP receptors.Results:In vitro and in vivo tests in rodents revealed that the structural elements of the wrist epitope combined with finger 2 and a positive charge proximal to the tip of this finger are sufficient to induce osteoinhibition with deletion mutants and folding variants of mature BMP‐4. The inhibitor designed to suppress heterotopic ossification showed BMP antagonist activity in embryos and animal caps of X. laevis. Binding studies of the inhibitor to ectodomains of type I and type II BMP receptors revealed a concentration‐dependent binding, especially to the high‐affinity BMP receptor.Conclusions:Deletion mutants of BMP folding variants are a new form of BMP antagonists and act through competition with osteoinductive BMP for BMP receptor binding. The excellent in vivo performance of the optimized folding variant is because of its ability to block signaling of endogenous BMPs deposited in the extracellular matrix even more effectively than the natural occurring BMP antagonist Noggin.

Heterotopic ossification is a frequent complication in patients who have suffered head and neck traumas or have undergone total hip replacement. In this report, stable folding variants of the natural occurring osteoinductive BMPs were shown to act as inhibitors for heterotopic ossification. The most effective BMP folding variant construct performed even better than the natural occurring BMP antagonist Noggin because it also inhibited calcium deposition of pre‐osteoblastic cells.

Heterotopic ossification is a frequent complication in patients who have suffered head and neck traumas or undergone total hip replacement. Heterotopic ossification occurs when osteogenic precursor cells present at the ectopic site receive the necessary signal(s) to differentiate into osteoblasts. At the protein level, the key factors in differentiation of cells to the osteogenic lineage are BMPs. Stable BMP variants derived from the identical amino acid sequence but with different disulfide bridge configurations have been investigated and found to be capable of inhibiting ossification in vitro and in vivo in rodents. These findings provide a concept for the straightforward development of a novel class of BMP antagonists that could lead to new treatments for traumatically and genetically induced heterotopic ossification and also, possibly, for disorders in which other members of the TGF-β superfamily are involved.

Subjecting rabbit small intestinal brush border membrane vesicles (BBMV) to freeze-thaw cycles releases water-soluble lipid exchange (transfer) proteins into the supernatant. They differ widely in apparent molecular weight and catalyze cholesterol, phosphatidylcholine, and phosphatidylinositol exchange between two populations of small unilamellar lipid vesicles. In order to determine their interrelations, the smallest water-soluble lipid exchange protein was purified to homogeneity by gel filtration on Sephadex G-75 and cation exchange chromatography on Mono S. It is a basic protein of apparent molecular mass of 13 ± 0.5 kDa. The purified protein was used to raise polyclonal antibodies. Polyclonal antibodies were also produced against a lipid exchange protein of apparent molecular mass of 100-120 kDa. By comparing lipid exchange, lipid binding, and immunological properties of the water-soluble lipid exchange proteins it can be shown that the 13-kDa (peak 3) protein is related to the 100-120 kDa (peak 1) protein; the properties of these two proteins are different from those of the peak 2 lipid exchange protein of apparent molecular mass of 22 kDa. Based on the immunological cross-reactivity observed between the 13 and 100-120 kDa and the lipid binding properties of these two proteins, a working hypothesis is proposed: both proteins are probably part of an intergral membrane protein of the brush border membrane that facilitates cholesterol and phosphatidylcholine absorption in this membrane. Evidence derived from immunogold labeling of BBMV supports the notion that this protein is located on the external (luminal) side of the brush border membrane. The analogous behavior of rabbit and human small intestinal brush border membrane in terms of lipid absorption and the release of water-soluble lipid exchange proteins is discussed.

Vertebrate striated muscle contains a set of myosin-associated proteins with discrete distributions in the A-band. Some of these proteins, including MyBP-H and MyBP-C, are characterized by a series of internal repeats (motifs) with homology to either the C2-set of the immunoglobulin superfamily or the fibronectin type III repeat. These repeats are predicted to be involved in protein--protein interactions within the myofibril. The cDNA sequence, the genomic organization, and the chromosomal localization of the human homologue of MyBP-H are presented. The 1.8-kb cDNA encodes a 52-kDa polypeptide containing two Ig-C2 and two type III repeats. The mRNA is expressed in a skeletal muscle-specific pattern. A 28-kb region of genomic sequence has been isolated that encompasses the 5′ and 3′ ends of the cDNA. This region includes 4.2 kb of upstream sequence with a potential promoter and 14 kb of downstream sequence containing the polyadenylation site. The chromosomal assignment was made by high resolution chromosomal in situhybridization. This method maps the gene to chromosome 1q32.1. The repeat structure described previously in chicken MyBP-H and MyBP-C was also detected in human MyBP-H. The primary sequence of the C-terminal Ig-C2 motif and its predicted secondary structure have been extensively conserved in MyBP-H homologues and other members of the MyBP family. This Ig-C2 motif has been implicated in myosin binding.

The uptake of free and esterified cholesterol at the brush border membrane is protein‐mediated. Here we show that this sterol uptake is effectively inhibited by exchangeable serum apolipoproteins. Binding of the apolipoprotein to the brush border membrane mediates the inhibitory effect. Evidence is presented to show that the structural motif responsible for the inhibition is the amphipathic α‐helix.

An up to 14‐fold increase in total hexokinase activity induced by low‐frequency stimulation in rat fast‐twitch muscle was followed by a rapid decay in enzyme activity after cessation of stimulation. In vivo labeling revealed that these alterations were related to rapid changes in [35S]methionine incorporation into hexokinase II. A recovery period of 15 h after cessation of stimulation was sufficient to normalize the approximately 30‐fold elevated [35S]methionine incorporation.

An up to 14-fold increase in total hexokinase activity induced by low-frequency stimulation in rat fast-twitch muscle was followed by a rapid decay in enzyme activity after cessation of stimulation. In vivo labeling revealed that these alterations were related to rapid changes in [ 35S]methionine incorporation into hexokinase II. A recovery period of 15 h after cessation of stimulation was sufficient to normalize the approximately 30-fold elevated [ 35S]methionine incorporation.