Ectopic Endochondral Bone Formation Research Articles

Selective Retinoic Acid Receptor γ Antagonist 7C is a Potent Enhancer of BMP-Induced Ectopic Endochondral Bone Formation.

Bone morphogenetic proteins (BMPs) have been clinically applied for induction of bone formation in musculoskeletal disorders such as critical-sized bone defects, nonunions, and spinal fusion surgeries. However, the use of supraphysiological doses of BMP caused adverse events, which were sometimes life-threatening. Therefore, safer treatment strategies for bone regeneration have been sought for decades. Systemic administration of a potent selective antagonist of retinoic acid nuclear receptor gamma (RARγ) (7C) stimulated BMP-induced ectopic bone formation. In this study, we developed 7C-loaded poly lactic nanoparticles (7C-NPs) and examined whether local application of 7C enhances BMP-induced bone regeneration. The collagen sponge discs that absorbed recombinant human (rh) BMP-2 were implanted into the dorsal fascia of young adult mice to induce ectopic bone. The combination of rhBMP-2 and 7C-NP markedly increased the total bone volume and thickness of the bone shell of the ectopic bone in a dose-dependent manner compared to those with rhBMP-2 only. 7C stimulated sulfated proteoglycan production, expression of chondrogenic marker genes, and Sox9 reporter activity in both chondrogenic cells and MSCs. The findings suggest that selective RARγ antagonist 7C or the related compounds potentiate the bone inductive ability of rhBMP-2, as well as support any future research to improve the BMP-2 based bone regeneration procedures in a safe and efficient manner.

Fibrodysplasia Ossificans Progressiva (FOP): A Segmental Progeroid Syndrome.

Segmental progeroid syndromes are commonly represented by genetic conditions which recapitulate aspects of physiological aging by similar, disparate, or unknown mechanisms. Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disease caused by mutations in the gene for ACVR1/ALK2 encoding Activin A receptor type I/Activin-like kinase 2, a bone morphogenetic protein (BMP) type I receptor, and results in the formation of extra-skeletal ossification and a constellation of others features, many of which resemble accelerated aging. The median estimated lifespan of individuals with FOP is approximately 56 years of age. Characteristics of precocious aging in FOP include both those that are related to dysregulated BMP signaling as well as those secondary to early immobilization. Progeroid features that may primarily be associated with mutations in ACVR1 include osteoarthritis, hearing loss, alopecia, subcutaneous lipodystrophy, myelination defects, heightened inflammation, menstrual abnormalities, and perhaps nephrolithiasis. Progeroid features that may secondarily be related to immobilization from progressive heterotopic ossification include decreased vital capacity, osteoporosis, fractures, sarcopenia, and predisposition to respiratory infections. Some manifestations of precocious aging may be attributed to both primary and secondary effects of FOP. At the level of lesion formation in FOP, soft tissue injury resulting in hypoxia, cell damage, and inflammation may lead to the accumulation of senescent cells as in aged tissue. Production of Activin A, platelet-derived growth factor, metalloproteinases, interleukin 6, and other inflammatory cytokines as part of the senescence-associated secretory phenotype could conceivably mediate the initial signaling cascade that results in the intense fibroproliferative response as well as the tissue-resident stem cell reprogramming leading up to ectopic endochondral bone formation. Consideration of FOP as a segmental progeroid syndrome offers a unique perspective into potential mechanisms of normal aging and may also provide insight for identification of new targets for therapeutic interventions in FOP.

Tgfbr2 is required in Acan-expressing cells for maintenance of the intervertebral and sternocostal joints.

BackgroundMembers of the transforming growth factor beta (TGF‐β) family are secreted proteins that regulate skeletal development. TGF‐β signaling is critical in embryonic development of the annulus fibrosus (AF) of the intervertebral disc (IVD). To address the question of the role of TGF‐β signaling in postnatal development and maintenance of the skeleton, we generated mice in which Tgfbr2 was deleted at 2‐weeks of age in Aggrecan (Acan)‐expressing cells using inducible Cre/LoxP recombination.MethodsLocalization of Cre recombination was visualized by crossing Acantm1(cre/ERT2)Crm mice to fluorescent mTmG reporter mice. Acantm1(cre/ERT2)Crm mice were mated to Tgfbr2 LoxP/LoxP mice and Cre recombinase was activated by tamoxifen injection at 2‐weeks postnatally. Following tamoxifen injection, mice were aged to 3, 6, and 12‐months and control mice were compared to the experimental (cKO) group. Mice were initially analyzed using X‐ray and skeletal preparations. Sternocostal joints and IVD tissues were further analyzed histologically by hematoxylin and eosin (H&E), Safranin O, and Picrosirius Red staining as well as Col10 immunostaining.ResultsCre recombination was observed in the IVD and sternocostal joints. X‐ray analysis revealed osteophyte formation within the disc space of 12‐month‐old cKO mice. Skeletal preparations confirmed calcification within the IVD and the sternocostal joints in cKO mice. H&E staining of cKO IVD revealed disorganized growth plates, delay in the formation of the bony endplate, and Col10 staining in the AF indicative of ectopic endochondral bone formation. Furthermore, proteoglycan loss was observed and collagen bundles within the inner AF were thinner and less organized. Alterations in the IVD were apparent beginning at 3 months and were progressively more visible at 6 and 12 months. Similarly, histological analysis of cKO sternocostal joints revealed joint calcification, proteoglycan loss, and disorganization of the collagen architecture at 12 months of age.ConclusionsTGF‐β signaling is important for postnatal development and maintenance of fibrocartilaginous IVD and sternocostal joints.

The fibrodysplasia ossificans progressiva R206H ACVR1 mutation activates BMP-independent chondrogenesis and zebrafish embryo ventralization

Patients with classic fibrodysplasia ossificans progressiva, a disorder characterized by extensive extraskeletal endochondral bone formation, share a recurrent mutation (R206H) within the glycine/serine-rich domain of ACVR1/ALK2, a bone morphogenetic protein type I receptor. Through a series of in vitro assays using several mammalian cell lines and chick limb bud micromass cultures, we determined that mutant R206H ACVR1 activated BMP signaling in the absence of BMP ligand and mediated BMP-independent chondrogenesis that was enhanced by BMP. We further investigated the interaction of mutant R206H ACVR1 with FKBP1A, a glycine/serine domain-binding protein that prevents leaky BMP type I receptor activation in the absence of ligand. The mutant protein exhibited reduced binding to FKBP1A in COS-7 simian kidney cell line assays, suggesting that increased BMP pathway activity in COS-7 cells with R206H ACVR1 is due, at least in part, to decreased binding of this inhibitory factor. Consistent with these findings, in vivo analyses of zebrafish embryos showed BMP-independent hyperactivation of BMP signaling in response to the R206H mutant, resulting in increased embryonic ventralization. These data support the conclusion that the mutant R206H ACVR1 receptor in FOP patients is an activating mutation that induces BMP signaling in a BMP-independent and BMP-responsive manner to promote chondrogenesis, consistent with the ectopic endochondral bone formation in these patients.

The Dose of Growth Factors Influences the Synergistic Effect of Vascular Endothelial Growth Factor on Bone Morphogenetic Protein 4–Induced Ectopic Bone Formation

Although vascular endothelial growth factor (VEGF) has been shown to act synergistically with bone morphogenetic protein (BMP)2 and BMP4 to promote ectopic endochondral bone formation via cell-based BMP gene therapy, the optimal ratio of VEGF to either of the BMPs required to obtain this beneficial effect remains unclear. In the current study, two cell types (C2C12, NIH/3T3) were retrovirally transduced to express BMP4 only or both BMP4 and VEGF. The resulting groups of cells were tested for their cellular proliferation, in vitro mineralization capacity, survival potential, and ability to undergo ectopic bone formation when implanted into a gluteofemoral muscle pocket created in severe combined immunodeficient mice. Results showed that VEGF inhibited the in vitro calcification of C2C12 and NIH/3T3 cells transduced to express BMP4. In vivo, C2C12 and NIH/3T3 cells expressing BMP4 and VEGF displayed significantly less bone formation than the same cells expressing only BMP4. In vivo, our results indicated that, when the ratio of VEGF to BMP4 is high, a detrimental effect on ectopic bone formation is observed; however, when the ratio is kept low and constant over time, the detrimental effect that VEGF has on ectopic bone formation is lost. Our studies revealed that VEGF's synergistic role in BMP4 induced ectopic bone formation is dose and cell-type dependent, which is an important consideration for cell-based gene therapy and tissue engineering for bone healing.

The Orphan Nuclear Receptor Estrogen Receptor-related Receptor γ Negatively Regulates BMP2-induced Osteoblast Differentiation and Bone Formation

Estrogen receptor-related receptor gamma (ERRgamma/ERR3/NR3B3) is a member of the orphan nuclear receptor with important functions in development and homeostasis. Recently it has been reported that ERRalpha is involved in osteoblast differentiation and bone formation. In the present study we examined the role of ERRgamma in osteoblast differentiation. Here, we showed that ERRgamma is expressed in osteoblast progenitors and primary osteoblasts, and its expression is increased temporarily by BMP2. Overexpression of ERRgamma reduced BMP2-induced alkaline phosphatase activity and osteocalcin production as well as calcified nodule formation, whereas inhibition of ERRgamma expression significantly enhanced BMP2-induced osteogenic differentiation and mineralization, suggesting that endogenous ERRgamma plays an important role in osteoblast differentiation. In addition, ERRgamma significantly repressed Runx2 transactivity on osteocalcin and bone sialoprotein promoters. We also observed that ERRgamma physically interacts with Runx2 in vitro and in vivo and competes with p300 to repress Runx2 transactivity. Notably, intramuscular injection of ERRgamma strongly inhibited BMP2-induced ectopic bone formation in a dose-dependent manner. Taken together, these results suggest that ERRgamma is a novel negative regulator of osteoblast differentiation and bone formation via its regulation of Runx2 transactivity.

Wnt pathway, an essential role in bone regeneration

Fracture repair is a complex regenerative process initiated in response to injury, resulting in optimal restoration of skeletal function. Although histology characteristics at various phases of fracture repair are clear and well established, much remains to be understood about the process of bone healing, particularly at the molecular signaling level. During the past decade, secreted signaling molecules of the Wnt family have been widely investigated and found to play a central role in controlling embryonic development processes. Wnt signaling pathway also plays a pivotal role in the regulation of bone mass. Recent published data reveal that Wnt signaling pathway is activated during postnatal bone regenerative events, such as ectopic endochondral bone formation and fracture repair. Dysregulation of this pathway greatly inhibits bone formation and healing process. Interestingly, activation of Wnt pathway has potential to improve bone healing, but only utilized after mesenchymal cells have become committed to the osteoblast lineage. These advances suggest an essential role of Wnt pathway in bone regeneration.

BMP type I receptor inhibition reduces heterotopic [corrected] ossification.

Fibrodysplasia ossificans progressiva (FOP) is a congenital disorder of progressive and widespread postnatal ossification of soft tissues and is without known effective treatments. Affected individuals harbor conserved mutations in the ACVR1 gene that are thought to cause constitutive activation of the bone morphogenetic protein (BMP) type I receptor, activin receptor-like kinase-2 (ALK2). Here we show that intramuscular expression in the mouse of an inducible transgene encoding constitutively active ALK2 (caALK2), resulting from a glutamine to aspartic acid change at amino acid position 207, leads to ectopic endochondral bone formation, joint fusion and functional impairment, thus phenocopying key aspects of human FOP. A selective inhibitor of BMP type I receptor kinases, LDN-193189 (ref. 6), inhibits activation of the BMP signaling effectors SMAD1, SMAD5 and SMAD8 in tissues expressing caALK2 induced by adenovirus specifying Cre (Ad.Cre). This treatment resulted in a reduction in ectopic ossification and functional impairment. In contrast to localized induction of caALK2 by Ad.Cre (which entails inflammation), global postnatal expression of caALK2 (induced without the use of Ad.Cre and thus without inflammation) does not lead to ectopic ossification. However, if in this context an inflammatory stimulus was provided with a control adenovirus, ectopic bone formation was induced. Like LDN-193189, corticosteroid inhibits ossification in Ad.Cre-injected mutant mice, suggesting caALK2 expression and an inflammatory milieu are both required for the development of ectopic ossification in this model. These results support the role of dysregulated ALK2 kinase activity in the pathogenesis of FOP and suggest that small molecule inhibition of BMP type I receptor activity may be useful in treating FOP and heterotopic ossification syndromes associated with excessive BMP signaling.

Differential Effect of BMP4 on NIH/3T3 and C2C12 Cells: Implications for Endochondral Bone Formation

After intramuscular implantation, BMP4-expressing NIH/3T3 fibroblasts and BMP4-expressing C2C12 myoblasts can promote ectopic cartilage and bone formation. Fibroblasts tend to undergo chondrogenesis, whereas myoblasts primarily undergo osteogenesis. These results suggest that endochondral bone formation may involve different cell types, a finding that could have major implications for the tissue engineering of bone and cartilage. The delivery of BMP4 through cell-based gene therapy can trigger ectopic endochondral bone formation in skeletal muscle. We hypothesized that, when stimulated with or transduced to express BMP4, different types of cells residing within skeletal muscle might participate in different stages of endochondral bone formation. We compared the responses of a fibroblast cell line (NIH/3T3), a myoblast cell line (C2C12), primary fibroblasts, and primary myoblasts to BMP4 stimulation in vitro. We then transduced the four cell populations to express BMP4 and compared their ability to promote ectopic endochondral bone formation in skeletal muscle. Under the influence of BMP4 in vitro and in vivo, NIH/3T3 cells differentiated toward both chondrogenic and osteogenic lineages, whereas most C2C12 cells underwent primarily osteogenic differentiation. NIH/3T3 cells genetically modified to express BMP4 induced delayed but more robust cartilage formation than did genetically modified C2C12 cells, which promoted rapid ossification. These differences in terms of the timing and amount of cartilage and bone formation persisted even after we introduced a retrovirus encoding dominant negative Runx2 (DNRunx2) into the C2C12 cells, which interferes with the function of Runx2. Superior osteogenic potential was also displayed by the primary myoblasts in vitro and in vivo compared with the primary fibroblasts. The different proliferation abilities and differentiation potentials exhibited by these cells when influenced by BMP4 may at least partially explain the differing roles that BMP4-expressing myogenic cells and BMP4-expressing fibroblastic cells play in endochondral bone formation. Our findings suggest that the process of endochondral bone formation in skeletal muscle after delivery of BMP4 involves different cell types, including fibroblastic cells, which are more involved in the chondrogenic phases, and myoblastic cells, which are primarily involved in osteogenesis. These findings could have important implications for the development of tissue engineering applications focused on bone and cartilage repair.

DBM induced ectopic bone formation in the rat: The importance of surface area

Demineralized bone matrix (DBM) has been shown to induce ectopic endochondral bone formation, when intramuscularly implanted in rats. In earlier studies we have found a variation in bone formation capacity of this DBM. This might be due to the properties of the DBM itself, but the use of DBM blocks could be of influence as well. Therefore, this study was designed to investigate whether increasing the surface area of the DBM by morsellizing, influences the bone formation capacity. In view of this, DBM implants and morsellized DBM (MDBM) implants were placed intramuscularly in a rat model. At six weeks the implants were retrieved and evaluated by histology and histomorphometry. The results demonstrated that significant amounts of newly formed bone were present in some DBM as well as some MDBM implants while in others no, or very little new bone was found. Histomorphometric analysis showed an average bone formation of 2.6% in DBM implants and an average of 1.9% in MDBM implants. Still, the amount of bone formation was limited compared with previous studies. It is concluded that enlargement of the surface area by morsellizing DBM implants is not an important factor in bone forming capacity.

Establishment of a clonal human mesenchymal cell line that retains multilineage differentiation capacity from a spinal hamartoma.

We isolated a single-cell-derived cell line from a spinal hamartoma, a which occurred in a newborn boy and was associated with a rudimentary limb. The maternal cells (HHC-7) differentiated into osteoblasts, chondrocytes, adipocytes, and skeletal muscles when they were cultured in differentiation-inducing media specific to each mesenchymal cell. We isolated a single-cell-derived clonal cell line (Clone K) after transfection with SV40 T antigen. These cells expressed CD73 and CD117, while being negative for expression of CD45. Clone K cells cultured in an osteogenic differentiation medium increased ALP activity and expressed mRNAs for Runx2 and osteocalcin. Treatment with rhBMP-2 induced Clone K cells to differentiate into both osteoblasts and chondrocytes. These cells expressed mRNAs for Sox9 and aggrecan in addition to osteogenic markers. Culture in an adipogenic differentiation medium induced Clone K cells to differentiation into adipocytes, which expressed mRNAs for PPARgamma2 and a2P. Clone K cells cultured in a serum-depleted medium generated desmin-positive cells and expressed MyoD1 mRNA. Clone K cells exhibited numerous alpha-smooth muscle actin-positive cells; however, treatment with rhBMP-2 decreased their number. Clone K cells, transplanted with a carrier containing rhBMP-2 into the muscles of SCID mice, generated ectopic endochondral bone formation. In these tissues, several osteoblasts and chondrocytes expressed SV40 T antigen, indicating their Clone K cell origin. Thus, Clone K cells are useful tools for analyzing the characteristics of human multipotential mesenchymal progenitors.

The functional equivalence of demineralized bone and tooth matrices in ectopic bone induction.

The objective of this study was to determine whether demineralized rat incisor matrices were a more potent inducer of ectopic endochondral bone formation than demineralized diaphyseal bone matrices derived from the same donors. Twenty-five-milligram disks of demineralized bone or tooth matrix obtained from adolescent Long-Evans rats were implanted in a standardized ectopic site. Biochemical and histometric measurements of bone formation revealed that the two matrices were functionally equivalent inducers of endochondral bone formation. The induced pellicle of bone reached a maturation point 18 days after implantation. Dentin matrix implants generated a significantly greater amount of mineralized tissue than did bone matrix implants. This difference could be explained on the basis of remineralization of the dentin particles to a greater degree than the bone matrix particles. Initial observations suggesting a more robust osteoinductive activity in demineralized incisor matrix can be attributed to the decreasing activity of bone matrix from older donors when compared to younger donors. The extent of osteoinduction by the two substrata was equivalent when the matrices were matched for age.

Purification of bone morphogenetic protein and investigation of its effects on osteoblastic cell line UMR108

It is well known that the bone matrix contains proteins which can induce ectopic endochondral bone formation in vivo. One class of these proteins is the bone morphogenetic protein (BMP). In order to investigate the physiological function of the BMP, its purification was attempted from an extract of demineralized bone matrix and its actions on the osteoblastic cell line were investigated. To isolate the BMP, a demineralized bone matrix was extracted with 4M guanidine-HCl. A water-insoluble fraction (G-WI) was separated from the demineralized bone extract by dialysis against distilled water and centrifugation. The BMP was purified from G-WI by gel filtration on Sephacryl S-200 HR, cation exchange with Mono-S, heparin affinity column and finally by C1/8 reverse phase chromatography. Peptide sequence analysis revealed that the purified BMP fraction contained "BMP-3" reported by Wozney et al. (1988). In order to investigate its function, the BMP was applied to the rat osteogenic sarcoma cell line UMR108. The BMP inhibited the growth of the UMR108 cells and enhanced the alkaline phosphatase activity in a dose-responsive manner.

Accelerated endochondral osteoinduction in the absence of bone matrix particles in a rat model system

Ethanol-precipitated proteins obtained from demineralized rat bone powder (DBP) by 4M guanidine-HCl extraction have been shown to reproducibly induce ectopic endochondral bone formation when subcutaneously implanted in rats in the absence of bone matrix particles. Histologic and biochemical analysis revealed a temporal sequence of chondrocyte differentiation, calcified cartilage formation, neovascularization, osteoblast differentiation, bone formation, osteoclastic bone remodeling, and hematopoietic marrow development that is complete by 21 days. In contrast to previous reports, these results clearly show an osteoinductive response independent of the presence of insoluble extracellular bone matrix. Compared with conventional DBP implants, the guanidine-extractable protein (GE) produces an accelerated and more robust osteoinductive response. Histologically, the initial chondrogenic response at days 6 to 9 is greatly amplified. Alkaline phosphatase specific activity peaks at day 9, several days earlier than for DBP, and is sixfold higher. Calcium accumulation in GE implants at day 12 is fivefold greater than with DBP, and all mineral is localized within the matrix of newly calcified cartilage and new bone. Osteoclasts are up to ninefold more abundant in the rapidly remodeling GE ossicle, making space for hematopoietic marrow. Delivery of GE coprecipitated with inert bone matrix particles was also more effective than DBP, although the response was somewhat attenuated compared with GE alone. Bony filling of 4-mm defects in rat mandibular rami was elicited by 10 mg of GE and followed an endochondral process with increased neovascularization compared with DBP and unimplanted controls. This guanidine-extractable protein fraction should prove useful for inducing quantities of chondrocytes and osteoclasts for in vitro study, and for analysis of osteoinductive requirements. Likewise, ethanol-precipitated osteoinductive proteins implanted alone or with carrier particles hold promise for use in reconstructive surgery.

Influence of parathyroidectomy, 1,25-dihydroxyvitamin D 3 and high dietary calcium intake on demineralized bone matrix powder-induced bone formation in the rat

Demineralized bone matrix induces ectopic endochondral bone formation. We used this model to study the effect of parathyroidectomy (PTX), 1,25-dihydroxyvitamin D 3 treatment, and calcium enriched diet on bone formation in the rat. Hypocalcemia and hyperphosphatemia in PTX rats were corrected by 1,25-dihydroxyvitamin D 3 treatment (2 × 12.5 ng/day) or by calcium enriched diet (3% calcium). Serum 1,25-dihydroxyvitamin D 3 concentration was decreased in PTX rats and in intact rats with high dietary calcium intake. Calcium content of ectopic new bones (42 days after bone matrix implantation) was reduced in PTX rats compared with intact control rats. This could be prevented by 1,25-dihydroxyvitamin D 3 treatment. In contrast, calcium enriched diet led to diminished mineralization of ectopic bones both in intact and PTX rats. We conclude that the effect of parathyroidectomy on bone formation may be mediated by 1,25-dihydroxyvitamin D 3. 1,25-Dihydroxyvitamin D 3 directly stimulates bone formation in this model and this effect is not simply the result of increasing serum calcium concentration.

The Role of Cells Versus Matrix in Bone Induction

Earlier observations indicated that epithelial cells of urinary bladder, and transformed epithelial cells from human amnion (FL), epidermal carcinoma (HeLa), etc. can induce ectopic endochondral bone formation when implanted into the skeletal muscle of immunosuppressed or autologous animals. Such epithelial cells are associated with little matrix. Bone-inducing activity was also demonstrated in cultured osteosarcoma cells of murine and human origin or extracts thereof, and it is notable that these bone-inducing osteosarcoma cells grow in vitro with little matrix production. Finally, electron microscopy of in vitro cartilage induction showed that decalcified rodent bone that had been extensively extracted to remove cells still contained devitalized cells and cell fragments some of which made contact with inducible, cartilage-forming mesenchymal cells that had migrated in from cocultured muscle. Suggestions: These observations suggest that: 1) the bone-inducing agent(s) of both epithelial and mesenchymal cells may reside mostly in cells rather than matrix. Thus it may be premature to assume that bone "matrix" is the major source of bone-inducing agent in decalcified bone until the osteoinductive activity of residual bone cells has been assessed; and 2) that the osteoinductive agent, whether residing in epithelial cells or bone cells, may be the same or a similar factor operating through the same mechanism.

Inhibition by tumor necrosis factor of induction of ectopic bone formation by osteosarcoma-derived bone-inducing substance

The effect of tumor necrosis factor alpha (TNF-α) on ectopic endochondral bone formation was studied in an experimental system for bone induction using murine osteosarcoma-derived bone-inducing substance. Ectopic new bone formation was inhibited by daily administration of recombinant human TNF-α (20–200 μg/kg body weight per day, intraperitoneally) after subcutaneous implantation of the bone-inducing substance into mice. Histological examination revealed that TNF-α prevented mesenchymal cells from differentiating into chondrocytes in the process of endochondral bone formation. The inhibitory effect of TNF-α continued during the period of its administration, but not after its administration was stopped. The bone induced in a three week period after discontinuation of TNF-α administration was histologically normal, but smaller than that induced in control animals. These findings suggested that TNF-α reversibly inhibits the biological activity of the bone-inducing substance or impairs the ability of cells to respond to the bone-inducing substance at an early stage of ectopic bone formation.

Partial isolation and characterization of a chemotactic factor from adult bovine bone for mesenchymal cells

Demineralized adult bone matrix has the capacity to initiate de novo ectopic endochondral bone formation 2–3 weeks following intramuscular implantation into suitable hosts. An early step in this process is the migration of mesenchymal cells to the implant site; these cells later differentiate into cartilage and bone. Adult bone has been shown to contain a number of bioactive factors, such as chemotactic factors for various cell types, including osteoblasts. We have used embryonic chick limb bud mesenchymal cells to construct an in vitro assay for testing chemotactic activity derived from bone matrix extracts. With a modified Boyden chamber, water-soluble components from a 4 M guanidinium chloride extract of demineralized adult bovine bone matrix were found to stimulate the directional migration of these chick embryonic limb bud mesenchymal cells as well as embryonic muscle-derived fibroblasts and cells derived from embryonic skin. The chemotactic activity was destroyed by treatment with heat (100 °C) or trypsin. Partial purification by molecular sieve chromatography suggested that the chemotactic factor(s) has a molecular weight of between 50,000 and 90,000. This factor can be separated from bone matrixderived chondrogenic stimulating activity by either ion exchange or molecular sieve chromatography. These observations confirm that bone matrix contains a chemoattractant for mesenchymal cells that may be important for in vivo recruitment of cells as part of the process of ectopic bone formation or in cases of bone repair.