Marshall R. Urist

Bone: Formation by Autoinduction

Wandering histiocytes, foreign body giant cells, and inflammatory connective-tissue cells are stimulated by degradation products of dead matrix to grow in and repopulate the area of an implant of decalcified bone. Histiocytes are more numerous than any other cell form and may transfer collagenolytic activity to the substrate to cause dissolution of the matrix. The process is followed immediately by new-bone formation by autoinduction in which both the inductor cells and the induced cells are derived from ingrowing cells of the host bed. The inductor cell is a descendant of a wandering histiocyte; the induced cell is a fixed histiocyte or perivascular young connective-tissue cell. Differentiation of the osteoprogenitor cell is elicited by local alterations in cell metabolic cycles that are as yet uncharacterized.

Matrix Gla protein, a new γ-carboxyglutamic acid-containing protein which is associated with the organic matrix of bone

A new protein has been isolated from CaCl2/urea extracts of demineralized bovine bone matrix. This protein has five to six residues of the vitamin K-dependent amino acid, gamma-carboxyglutamic acid (Gla), and we have accordingly designated it matrix Gla protein. Matrix Gla protein is a 15,000 dalton protein whose amino acid composition includes a single disulfide bond. The absence of 4-hydroxyproline in matrix Gla protein demonstrates that it is not a precursor to bone Gla protein, 5,800 dalton protein which has a residue of 4-hydroxyproline at position 9 in its sequence. Matrix Gla protein also does not cross-react with antibodies raised against bone Gla protein.

The reaction of the dura to bone morphogenetic protein (BMP) in repair of skull defects.

Trephine defects in the adult rat skull 0.8 cm in diameter, which do not spontaneously heal, were filled with a bovine bone morphogenetic protein (BMP) fraction. The defects healed not only by bony ingrowth from the trephine rim, but also by proliferation of pervascular mesenchymal-type cells (pericytes) of the dura mater. Under the influence of BMP, dural pericytes differentiated into chondroid and woven bone. Between three and four weeks postimplantation, sinusoids formed and the woven bone remodelled into lamellar bone. Concurrently, blood-borne bone marrow cells colonized the bone deposits, and the diploe were restored. Demonstrating that it is soluble in interstitial fluid, and diffusible across a nucleopore membrane (which isolated the bony margins of the skull), BMP induced new bone formation in the underlying dura and complete repair of the defect. The response of the dura to the BMP fraction produced more new bone than the response to allogeneic bone matrix. The BMP-induced repair was dose dependent; the quantity of new bone was proportional to the dose of the implanted BMP.

Osteogenetic potency and new-bone formation by induction in transplants to the anterior chamber of the eye.

As has been indicated above, it is not always possible to determine whether bone formation, following transplantation of a tissue to a favorable environment, is the result of proliferation and osteogenetic activity of transplanted cells, or whether the cells of the host, under the influence of induction as a result of the transplant, are responsible for the appearance of new bone. In an attempt to resolve this difficulty and for the purposes of exposition, there is presented a diagram (Fig. 12) which illustrates the probability that one or both of the mechanisms are operating in any given instance. In the upper portion of the diagram are shown those tissues in which new-bone formation is almost certainly the result of the osteogenetic activity of the transplanted cells (tissue cultures, periosteum transplanted to the eye); in the lower portion are devitalized tissues which cannot conceivably give rise to new bone by proliferation, and which form one of the strongest arguments for the phenomenon of induction. Between these extremes are various tissues which may act by one or both mechanisms, and an attempt has been made to arrange these in some semblance of order to illustrate the probabilities in the case of each tissue. At the middle of the scale it appears highly probable that cancellous bone and fibrocartilaginous callus may proliferate with formation of new bone and also induce bone formation by their effects upon the cells of the host. There is thus proposed a resolution of a basic problem of osteogenesis by acceptance of the postulate that post-foetal new-bone formation, instead of depending solely upon one of the two mechanisms which have been proposed, may occur through the effect of either of these mechanisms, or by a combination of the two.

Resistant nonunions and partial or complete segmental defects of long bones. Treatment with implants of a composite of human bone morphogenetic protein (BMP) and autolyzed, antigen-extracted, allogeneic (AAA) bone.

Twenty-five patients with resistant nonunions including partial or complete segmental defects were treated with a composite alloimplant of human bone morphogenetic protein (h-BMP) and autolyzed, antigen-free, allogeneic bone (AAA). The series consisted of 16 females and nine males; average age was 45 years. Preoperative symptoms averaged 30 months (range, five to 83 months); 22 of 25 patients had failed multiple attempts at electrical stimulation. Twenty-three of 25 patients had an average of three prior failed surgical attempts at union (range, one to ten). There were ten segmental defects with an average length of 4 cm (range, 2-9 cm). The composite implant was incorporated as an onlay in 15 extremities and as an inlay graft supported by internal fixation in ten extremities. Seven patients received supplementary autogeneic cancellous bone grafting. Average healing time was six months (range, three to 14 months). Average follow-up time was 21 months (range, five to 82 months). Functional results were rated as excellent, 14; good, five; and fair, five. One failed to unite because of a recurrent infection. Union was obtained in 24 of 25 patients. There were five failures of the original operation that required reoperations; union eventually occurred in four of five extremities by repeat composite grafting and replacement of the failed internal fixation. Bony union between host bone and the composite implant began at an average of eight weeks postoperatively. Present results indicate that h-BMP/AAA composite implants represent adjunctive treatment of difficult nonunions. The h-BMP/AAA composite implants may be implanted in either partial or complete segmental defects of long bones.(ABSTRACT TRUNCATED AT 250 WORDS)

Treatment of osteonecrosis of the femoral head with core decompression and human bone morphogenetic protein

A retrospective evaluation was done of 15 patients (17 hips) with symptomatic osteonecrosis of the hip treated with core decompression combined with an allogeneic, antigen-extracted, autolyzed fibula allograft and 50 mg of partially purified human bone morphogenetic protein and noncollagenous proteins. The average duration of clinical followup of the patients was 53 months (range, 26-94 months). The osteonecrotic involvement of the hip was classified by plain radiographs using a modification of the Ficat staging system and MRI evaluations. Fifteen hips were classified as Ficat Stage IIA, one hip (one patient) was classified as Ficat Stage IIB, and one hip (one patient) was classified as Ficat Stage III. Fourteen hips had involvement of 50% or less of the femoral head and 2/3 or less involvement of the weight-bearing surface of the femoral head, based on a magnetic resonance imaging evaluation. The procedures were a clinical success in 14 of 15 hips (93%; 13 patients) with Stage IIA disease. Three of 17 hips (three patients) had radiographic progression (Ficat Stages IIA, IIB, and III) of the femoral head and were converted to total hip replacements. Only one of seven hips (six patients) with 50% or less involvement of the femoral head and between 1/3 and 2/3 of the weightbearing surface of the femoral head developed radiographic progression of the femoral head. There was no radiographic progression in the 3 hips with less than 1/3 involvement of the weightbearing surface of the femoral head. Further evaluation of the potential efficacy of bone morphogenetic protein is required in randomized trials.

Beta-tricalcium phosphate delivery system for bone morphogenetic protein.

An aggregate of biodegradable beta-tricalcium phosphate and bone morphogenetic protein (BMP/TCP) induces the differentiation of cartilage within eight days, cartilage and woven bone within 12 days, and lamellar bone, including bone marrow, within 21 days. The yield of new bone from a 1-mg dose was more than 12 times greater from the TCP/BMP than from the BMP alone. Whether TCP acts as a slow-release delivery system, potentiates the activity of BMP, or serves to distribute BMP in a favorable three-dimensional pattern requires further investigation.

Human bone morphogenetic protein (hBMP)

Abstract Human bone morphogenetic protein (hBMP) was chemically extracted from demineralized gelatinized cortical bone matrix by means of a CaCl2·urea inorganic-organic solvent mixture, differential precipitation in guanidine hydrochloride, and preparative gel electrophoresis. hBMP is isolated in quantities of 1 mg/kg of wet weight of fresh bone, and has the amino-acid composition of an acidic polypeptide. The mol wt is 17 to 18 k-Da (kilodaltons). Implants of the isolated 17-kDa protein are very rapidly adsorbed and produce a smaller volume of bone than protein fractions consisting of 24-, 17-, and 14-kDa proteins. Since the isolated 24- and 14-kDA components lack hBMP activity, the kinetics of the bone morphogenetic processes including the function of other proteins as carrier molecules, await investigation.

Bone morphogenetic protein : The molecularization of skeletal system development

PROGRESS IN SCIENCE appears part and parcel with the introduction of new experimental methods. Three landmarks in the history of experimental embryology emerged from new methods. The first was von Baer’s experiments showing how the chick embryo develops from a more general to a specific, detailed body design. The second was Spemann’s microsurgical experiments on embryonic tissues showing the effects of one tissue upon another leading to differentiation of a third, and hypothetical chemical organizers of embryonic development. The third was in situ hybridization (Pardue et al., 1970) modified and improved by Harland for localization of bone morphogenetic protein (BMP) including BMP receptors. BMP is a hydrophobic low molecular weight polypeptide that evolved in species in the fossil record 500 million years ago. It is distinguished by heterotopic bone development in response to implants of nanogram quantities with a carrier in muscle. In this issue of JBMR, Nifuji et al. present circumstantial evidence of BMP-2– and -4–induced skeletal system development. The evidence is based on whole mounts of chick embryos and in situ hybridization, using digoxigeninlabeled mRNA probes to localize endogenous BMP-2 and BMP-4. Exogenous BMP (rhBMP-2) was implanted in pellets of glass in dorsal mesoderm. Transcripts for BMP-4 ligand and its receptors were expressed in dorsal ectoderm and mesoderm. The implants of exogenous BMP-4 and BMP-2 produced anomalies of vertebra, ribs, and scapulae. A basic assumption is that BMP generates form and controls the morphogenetic or predifferentiation stages of development. The cytodifferentiation phase delineates the time of differentiation of chondrocytes, adipocytes, osteocytes, and fibrocytes. Before the cytodifferentiation phase takes place, cells aggregate and proliferate in condensation centers. BMP concentration gradients arise in condensation centers. Nifuji et al. show that BMP-4 and BMP-2 expression occurs earlier than heretofore shown, and in reviewing the literature, they point out that previous research on skeletal cell condensation in the developing mammalian embryos appeared coincidental with the expression of BMP-5. The authors also point out that BMP-2 and BMP-4 are mammalian homologs of dpp and 60 A proteins associated with dorsoventral patterning in the fruit fly embryo, and of activan in the amphibian Xenopus. Similarly, in sea urchins, silkworms, and earthworms, to baboons and human beings, BMP or BMP antecedent molecules pervade the earliest stages of development of all animal life. BMPs have been observed in nearly all developing visceral and somatic organs, i.e., brain and sympathetic neurons, heart, liver, lung, skin, hair follicles, craniofacial structures, branchial arches, placenta, and skeletal elements. Improved methods of in situ hybridization and immunocytochemistry made it possible to colocalize BMP-2 to BMP-4 and two number I BMP receptors in various extraskeletal, paraskeletal, and skeletal tissues. Hogan proposed that, depending on the concentration, at low levels BMP would stimulate cell proliferation, whereas at high levels BMP might promote differentiation. In embryogenesis, BMPs appeared to be omnipresent, as essential as components of the cytoskeleton, i.e., actin, tublin, filament networks, vitamin D, binding protein, depactin, actophorin, villin, b-actinin, etc. Activin, inhibin, and retinoic acid are also closely associated with BMP and related proteins. In postfetal mammalian species, BMP-2 to BMP-7 are expressed in bone generation, regeneration, modeling, and remodeling of cartilage and bone, including ligament or tendon connective tissue insertions. In general, depending on concentration gradients and locations, BMP inhibits cell proliferation and induces cytodifferentiation. The history of BMP evolved from observations of allogeneic bone matrix–induced cartilage and bone development in mammalian species. The name stems from the demonstration of a hydrophobic noncollagenous glyco-

Repair of segmental defects of the tibia with cancellous bone grafts augmented with human bone morphogenetic protein: a preliminary report

Human bone morphogenetic protein (hBMP) is a bone cell differentiation-inducing factor. Six patients with traumatic segmental three- to 17-cm tibial defects developed solid union by implantation of hBMP and autogeneic cancellous grafts and stabilization. There were no allergic, infectious, or surgical complications. If hBMP augmentation in biodegradable delivery systems can be established by a prospective, randomized, double-blind investigation, the incidence of successful bone graft operations for treatment of large segmental defects would be measurably improved.