Bone Inductive Research Articles

The Induction of Bone Formation: The Translation Enigma.

A paradigmatic shift in the way of thinking is what bone tissue engineering science requires to decrypt the translation conundrum from animal models into human. The deductive work of Urist (1965), who discerned the principle of bone induction from the pioneering works of Senn, Huggins, Lacroix, Levander, and other bone regenerative scientists, provided the basis that has assisted future bone tissue regenerative scientists to extend the bone tissue engineering field and its potential uses for bone regenerative medicine in humans. However, major challenges remain that are preventing the formation of bone by induction clinically. Growing experimental evidence is indicating that bone inductive studies are non-translatable from animal models into a clinical environment. This is preventing bone tissue engineering from reaching the next phase in development. Countless studies are trying to discern how the formation of bone by induction functions mechanistically, so as to try and solve this enigmatic problem. However, are the correct questions being asked? Why do bone inductive animal studies not translate into humans? Why do bone induction principles not yield the same extent of bone formation as an autogenous bone graft? What are bone tissue engineering scientists missing? By critically re-assessing the past and present discoveries of the bone induction field, this review article attempts to re-discover the field of bone formation by induction, identifying some key features that may have been missed. These include a detailed library of all proteins in bones and their arrangement in the 3D superstructure of the bone together with some other important criteria not considered by tissue engineering scientists. The review therefore not only re-iterates possible avenues of research that need to be re-explored but also seeks to guide present and future scientists in how they assess their own research in light of experimental design and results. By addressing these issues bone formation by induction without autografts might finally become clinically viable.

EVALUATION OF THE OSTEOINDUCTIVE POTENTIAL OF FREEZE-DRIED HUMAN AMNIOTIC MEMBRANE (EXPERIMENTAL STUDY)

INTRODUCTION: Bone regeneration represents an important challenge in oral surgery. Several means have been employed to improve bonehealing, each having several advantages and disadvantages. The search for new materials and methods is an ongoing process.OBJECTIVES: The aim of the present study was to evaluate the bone inductive effect of the freeze-dried human amniotic membrane (FDAM)as a novel biomaterial for bone regeneration.MATERIALS AND METHODS: Twelve adult male dogs were involved in this study. Bilateral critical-sized mandibular defects were createdin each dog. One defect was left uncovered as a control. The other defect was covered with a double layered sterile freeze-dried human amnioticmembrane (FDAM) to serve as a study. At each of three time points - 4, 8, and 12 weeks - four dogs were euthanized and their mandibles wereharvested en bloc and osteotomy sites were submitted for histochemical examination to evaluate bone healing.RESULTS: The tissue samples were obtained after 4, 8, and 12 weeks for histochemical examination. The FDAM was found to enhance theblood supply to the defect area in the study group and gave rise to bone induction (P ˂0.001).CONCLUSIONS: Our study findings indicate that the FDAM has the potential for the enhancement of bone healing and bone induction.KEYWORDS: Amniotic membrane, freeze-dried, bone regeneration, critical-sized bone defect.

Ectopic Bone Induction by BMP-loaded Collagen Scaffold and Bone Marrow Stromal Cell Sheet

19 INTRODUCTION Bone inductive therapies have been developed using tissue engineering with three essential factors, i.e., osteogenic cells, osteoinductive growth factor and a scaffold for bone-forming cells. Bone marrow stromal cells (BMSCs) include multipotent mesenchymal stem cells. BMSCs could differentiate into several cell-types including osteoblasts , and implantation of BMSCs with various biological scaffolds enables to induce a new bone in vivo. Grafting with calcium alginate to alveolar bone defects in dogs accelerated bone regeneration. Bruder et al. reported that the combined implantation with hydroxyapatite/β-tricalcium phosphate ceramics and bone marrow-derived mesenchymal stem cells facilitated bone formation: They insisted that a ceramic scaffold carrying cell source could be primarily biocompatible and be able to provide mechanical stiffness compared to the ceramics scaffold per se. A biological scaffold may play an Ectopic Bone Induction by BMP-loaded Collagen Scaffold and Bone Marrow Stromal Cell Sheet

M644: Clinical Applications of Recombinant Human Bone Morphogenetic Protein-2 (rhBMP-2)

Human Bone Morphogenetic Proteins (BMP) are a group of bone inductive proteins that determine and form the human skeleton. In the adult, they reform bone after osteoclastic resorption as part of the normal bone turnover cycle. The concentration of BMP in human and animal bone is exceedingly small negating its clinical usefulness for bone induction via xenogenic or allogenic bone grafts. However, molecular biotechnology has cloned the BMP-2 genes to produce clinically effective concentrations in a recombinant form.

M632: Clinical Applications of Recombinant Bone Morphogenetic Protein-2 (rhBMP-2)

Human Bone Morphogenetic Proteins (BMP) are a group of bone inductive proteins that determine and form the human skeleton. In the adult, they reform bone after osteoclastic resorption as part of the normal bone turnover cycle. The concentration of BMP in human and animal bone is exceedingly small negating its clinical usefulness for bone induction via xenogenic or allogenic bone grafts. However, molecular biotechnology has cloned the BMP-2 genes to produce clinically effective concentrations in a recombinant form.

Repair of bone defects with rhBMP2 and a synthetic polymer

Titanium is a material with good bone affinity and bone inductive properties. We used it for new type of titanium implant to facilitate bone induction through BMPs and a carrier polymer. The carrier used for rhBMP-2 was poly-lactic acid-polyethyleneglycol block copolymar (PLA-PEG). Cylindrical titanium implants were formed with a porous surface saturated with the composite BMP/PLA-PEG. A bone defect was created in the humerus of a Japanese white rabbit, and the titanium/rhBMP-2/PLA-PEG composite was implanted with the nail fixation. After eight weeks, the defect in the humerus has been completely covered over with new bone tissue. New bone formation was also found inside the titanium pores. These results suggest that the BMP/PLA-PEG composite, with which porous titanium surface had been impregnated, exuded from that surface and caused the surface of the implant to become covered with new bone. This titanium/rhBMP/PLA-PEG implant promises to be effective in treating lager bone deficits.

Morphogenesis and tissue engineering of bone and cartilage: inductive signals, stem cells, and biomimetic biomaterials.

Morphogenesis is the developmental cascade of pattern formation, body plan establishment, and the architecture of mirror-image bilateral symmetry of many structures and asymmetry of some, culminating in the adult form. Tissue engineering is the emerging discipline of design and construction of spare parts for the human body to restore function based on principles of molecular developmental biology and morphogenesis governed by bioengineering. The three key ingredients for both morphogenesis and tissue engineering are inductive signals, responding stem cells, and the extracellular matrix. Among the many tissues in the human body, bone has considerable powers for regeneration and is a prototype model for tissue engineering based on morphogenesis. Implantation of demineralized bone matrix into subcutaneous sites results in local bone induction. This model mimics sequential limb morphogenesis and permitted the isolation of bone morphogens. Although it is traditional to study morphogenetic signals in embryos, bone morphogenetic proteins (BMPs), the inductive signals for bone, were isolated from demineralized bone matrix from adults. BMPs and related cartilage-derived morphogenetic proteins (CDMPs) initiate, promote, and maintain chondrogenesis and osteogenesis and have actions beyond bone. The symbiosis of bone inductive and conductive strategies are critical for tissue engineering, and is in turn governed by the context and biomechanics. The context is the microenvironment, consisting of extracellular matrix, which can be duplicated by biomimetic biomaterials such as collagens, hydroxyapatite, proteoglycans, and cell adhesion proteins including fibronectins. Thus, the rules of architecture for tissue engineering are an imitation of the laws of developmental biology and morphogenesis, and thus may be universal for all tissues, including bones and joints.

Evaluation of 2 novel approaches for assessing the ability of demineralized freeze-dried bone allograft to induce new bone formation.

Because of the wide variation in the ability of human demineralized freeze-dried bone allograft (DFDBA) to reproducibly induce new bone formation, there is a need for a reliable measure of bone induction activity. In this study we examined an immature osteoprogenitor cell line for its potential utility in measuring the activity of DFDBA in vitro. We characterized the response of 2T9 cells, an immature osteoprogenitor cell line derived from the calvariae of transgenic mice containing the SV40 T-antigen driven by the mouse bone morphogenetic protein (BMP)-2 promoter, to recombinant human BMP-2 by measuring alkaline phosphatase specific activity, osteocalcin production, and matrix mineralization. Responses were compared to those obtained with 1,25-(OH)2D3. In addition, 2T9 cells were cultured with active or inactive human DFDBA in the presence or absence of BMP-2. We also tested the hypothesis that radio-opacity of tissue following implantation of DFDBA in vivo correlates with the ability of human DFDBA to induce new bone. DFDBA from 9 different donors, stratified by age, were implanted subcutaneously in the thorax of 18 nude (nu/nu) mice. Tissue was harvested at 36 days postoperatively and examined histologically and biochemically for calcium and phosphorus uptake. 2T9 cells exhibited a dose- and time-dependent response to soluble BMP-2. Proliferation was decreased and alkaline phosphatase activity, osteocalcin production, and mineralized nodule formation were increased. The effects were dose- and time-dependent. Peak effects on alkaline phosphatase and osteocalcin were noted on day 8, whereas mineral deposition did not begin to occur until day 12. 1,25-(OH)2D3 did not regulate these effects unless used with BMP-2. When the cells were exposed to active or inactive DFDBA in the presence or absence of BMP-2, no effect on 2T9 cell differentiation was observed. This indicated that DFDBA released no soluble factors with bone inductive ability and that if any active factors were adsorbed to the DFDBA, they were inactivated. When DFDBA was implanted subcutaneously in the thorax of nude mice, there was no histologic evidence of new bone formation. However, there was a donor age-dependent decrease in Ca and P uptake of the implanted tissue, reflecting a donor age-dependent decrease in remineralization of DFDBA. These data indicate that cell culture assays like the one used in this study may not be appropriate indicators of bone induction ability by DFDBA since soluble factors may not be responsible for bone induction in vivo. Nonetheless, in vitro assays are still needed. While Ca and P uptake by DFDBA-implanted tissue in the present study correlated with the age-dependent decrease in bone induction at intramuscular sites in a previously reported study, these data show that early x-rays may actually detect remineralization and not new bone formation. Thus, assessment of bone induction ability may still depend on histologic analysis of animal models.

Ethene oxide and bone induction. Controversy remains.

There is controversy as to whether ethene oxide ("ethylene oxide", EO) sterilization destroys the bone-inducing capacity of demineralized bone matrix (DBM) or not. Correctly performed studies seem to support both opinions. Bone conductive properties of fresh frozen, defatted bone grafts are greatly impaired by EO sterilization, whereas purified inductive proteins resist EO. Studies showing destruction of osteoinductive capacity used nonpulverized DBM, whereas the others used powder. This could be the key to resolving the controversy, because if EO treatment reduces the cells' ability to penetrate a cortical graft and to reach inductive proteins inside it, it may appear noninductive after EO sterilization, even though BMP molecules may be intact. On the other hand, cells could easily penetrate the powder implants. We compared the effect of EO sterilization on the inductive capacity of demineralized cortical bone with that of DBM powder, using allogeneic material in rats. Cortical pieces lost all inductive capacity by EO sterilization, whereas the powder yielded a calcium content which was at best one fourth of the unsterilized. The concentrations of residual EO, ethene chlorohydrin and ethene glucol at implantation were far below approved levels. Another difference between studies is the humidity during EO treatment. In our hands, humidification reduced bone yield by half. In conclusion, EO sterilization may impair the biological performance of bone inductive implants by reducing cell penetration into bulk material. However, DBM powder, when correctly sterilized, also yielded scanty amounts of bone.

Growth and Morphogenetic Factors in Bone Induction: Role of Osteogenin and Related Bone Morphogenetic Proteins in Craniofacial and Periodontal Bone Repair

Bone has considerable potential for repair as illustrated by the phenomenon of fracture healing. Repair and regeneration of bone recapitulate the sequential stages of development. It is well known that demineralized bone matrix has the potential to induce new bone formation locally at a heterotopic site of implantation. The sequential development of bone is reminiscent of endochondral bone differentiation during bone development. The collagenous matrix-induced bone formation is a prototype model for matrix-cell interactions in vivo. The developmental cascade includes migration of progenitor cells by chemotaxis, attachment of cells through fibronectin, proliferation of mesenchymal cells, and differentiation of bone. The bone inductive protein, osteogenin, was isolated by heparin affinity chromatography. Osteogenin initiates new bone formation and is promoted by other growth factors. Recently, the genes for osteogenin and related bone morphogenetic proteins were cloned and expressed. Recombinant osteogenin is osteogenic in vivo. The future prospects for bone induction are bright, and this is an exciting frontier with applications in oral and orthopaedic surgery.

Parallels between development of embryonic and matrix‐induced endochondral bone

Endochondral bone formation can take place in the embryo, during fracture healing, or in postnatal animals after induction by implanted demineralized bone matrix. This matrix-induced bone formation recapitulates the embryonic sequence of bone formation morphologically and biochemically. The steps in bone formation in both systems include differentiation of cartilage from mesenchyme, cartilage maturation, invasion of the cartilage by blood vessels and marrow precursors, and formation of bone and bone marrow. Recently, bone inductive molecules from demineralized bone matrix have been purified, sequenced and produced as recombinant proteins. While there are similarities between bone development in the embryo and that after induction by these purified molecules, the molecules responsible for bone induction in the embryo have not yet been defined. Because of similarities between the two methods of bone formation, studies of bone induction by demineralized bone matrix may help to elucidate mechanisms of embryonic bone induction.

Monkey bone matrix induces bone formation in the athymic rat, but not in adult monkeys

Demineralized bone matrix from young and adult monkeys was implanted intramuscularly for 6 weeks in athymic rats and adult monkeys. Cartilage and bone induction was evaluated by histology and calcium content. In the athymic rat, most implants induced cartilage or bone. In the monkeys, cartilage was formed only on rare occasions and there was no sign of bone formation. We conclude that (a) adult monkey bone matrix contains bone inductive properties; (b) these properties are not sufficient to induce bone formation in adult monkey muscle sites.

Dose-dependent reduction of bone inductive properties by ethylene oxide

Sterilisation of demineralised bone matrix with ethylene oxide has been claimed to destroy the ability of bone matrix to induce new bone formation on intramuscular implantation. Other workers have routinely used ethylene oxide sterilised bone matrix for assays in rodents without detrimental effects. We studied the effects of various lengths of exposure to ethylene oxide gas, and found that bone induction properties are destroyed in a dose-dependent manner. After a short exposure, bone induction properties were moderately diminished. However, this short ethylene oxide treatment did not kill Bacillus subtilis spores. A sterilisation procedure that killed these spores rendered the implants incapable of bone-induction.

Dissociative extraction and partial purification of osteogenin, a bone inductive protein, from rat tooth matrix by heparin affinity chromatography

Implantation of demineralized tooth matrix in subcutaneous sites results in new bone formation locally. The osteoinductive activity of the tooth matrix was dissociatively extracted in 4.0 M guanidine hydrochloride and the residue was devoid of biologic activity. The bone inductive protein, osteogenin, was partially purified by heparin affinity chromatography. The heparin binding fraction initiated the bone differentiation cascade when implanted with guanidine extracted, inactive bone or tooth matrices. These results imply a cooperative interaction between the soluble osteogenin and collagenous substratum in bone induction.

骨形成因子からみたレーザーの骨修復促進効果の実験的検討

The authors applied lasers to the experiment of the bone induction by a bone inductive substance, bone morphogenetic protein (BMP), in the subcutaneous region of a mouse in order to evaluate the bone wound healing activation effect of the lasers. GaAlAs laser and He-Ne laser of mW level of the output power were used in this experiment. As the results the bone formation in the induced bone by BMP with exposure to the lasers was superior to that of the unlaced case. This result presumes that the lasers stimulate the host tissues to lead to activate the postfetal cytodifferentiation of the undifferentiated mesenchymal cells induced by BMP into bone tissue through endochondral ossification. However, it cannot be proved whether the lasers activated the activity of BMP in the basal bone tissue or not from the results of this experiment alone.

Bone Matrix-induced Local Bone Induction

The sequential cellular changes in the implants in response to collagenous bone matrix-induced local bone formation include: binding of fibronectin to matrix, chemotaxis and attachment of progenitor cells, proliferation and differentiation of progenitor cells into chondrocytes, and finally osteogenesis and marrow differentiation. The cellular origin of osteogenic proteins is not clear. The present study compares the osteogenic potential of demineralized rat and porcine bone matrix by dissociative extraction and reconstitution. Judging from the Sephacryl S-200 gel filtration profiles of the dissociative extracts of rat and porcine matrix, the latter appears to be smaller. Under identical experimental conditions, the rat chondrosarcoma and osteosarcoma were examined for chondrogenic and osteogenic properties and found to be devoid of inductive potential. It is noteworthy that gel filtration fractions of rat chondrosarcoma on Sepharose CL-6B are inhibitory to bone inductive potential of demineralized rat bone matrix.

Extracellular matrix proteins involved in bone induction are vitamin D dependent

Subcutaneous implantation of demineralized diaphyseal bone matrix into allogeneic rats results in local formation of cartilage and bone. However, implantation of demineralized bone matrix obtained from rachitic rats did not induce bone. Rachitic bone matix was therefore dissociatively extracted with 4 M guanidine HCl and then reconstituted with an inactive collagenous residue of control as carrier. Such reconstituted materials also lacked bone inductive potential. On the other hand, reconstitution of guanidine HCl extracts of control bone matrix with inactive vitamin D deficient matrix did result in bone induction. Partial purification (fractions containing proteins (< 50,000 daltons) of the guanidine HCl extract from rachitic rats on Sepharose CL-6B followed by reconstitution with inactive collagenous residues resulted in a weak (25% of control) inductive response. These observations imply that bone inductive proteins are vitamin D dependent and are reduced in matrix obtained from rachitic rats.

Distribution of bone inductive proteins in mineralized and demineralized extracellular matrix

Implantation of demineralized extracellular bone matrix results in new bone formation locally. Although the precise molecular mechanisms are not known, the reconstitution of matrix proteins less than 50,000 daltons with collagenous residue results in bone induction. The aim of the present investigation was to ascertain the distribution of the bone inductive protein(s) in various compartments of the tissue. A sequential extraction of mineralized bone matrix was employed: (1) 4 M guanidine HCl to extract proteins that are cell associated and not masked by mineral; (2) 0.5 M EDTA to dissolve the mineral phase; (3) 4 M guanidine HCl to reextract the collagenous matrix-associated proteins under dissociative conditions; (4) 4 M guanidine HCl containing 0.5 M EDTA to release any other residual proteins. This sequential method revealed that about 25% of total biological activity of bone induction is associated with first guanidine extraction, about 15% with the mineral phase and the rest of the activity is tightly associated with the collagenous matrix.

The Bone Inductive Capacity of Various Bone Transplanting Materials Used for Treatment of Experimental Bone Defects

The bone defect repairing capacities of autogenous cancellous bone (ACB), decalcified bone (DB), autodigested "low-antigenic" bone matrix (LBM) and preserved bone (PB) were tested in experimental defects in rabbit scapulae. The healing of the defects was most effective with ACB transplants as judged by roentgenologic and microscopic studies. The DB and LBM transplants caused states of healing comparable to each other, and the PB transplants showed the least satisfactory results in this experiment. The ACB, DB and LBM transplants caused bone induction, which resulted in the formation of new bone, subsequent resorption of the transplanted material and rapid reshaping of the callus. The PB transplants caused no bone induction; only some growth of callus on the edges of the defect was observed and pieces of the transplanted material were seen as late as 10 weeks after the operation as dead particles encapsulated by fibrous tissue. The infiltrations of inflammatory cells around the PB transplants indicated a considerable host versus graft reaction whereas this reaction was less with the other materials tested.