Connective Tissue Research, 50:1–6, 2009Copyright c© Informa Healthcare USA, Inc.ISSN: 0300-8207 print / 1521-0456 onlineDOI: 10.1080/03008200802690661

Original Research

Heterotopic Osteogenesis by Murine DemineralizedIncisors at Lesions Sites Induced by Concanavalin A in Mice

Paweł K. Włodarski, Ryszard Galus, and Krzysztof H. Włodarski

Department of Histology and Embryology, Center for Biostructure Research, Medical University ofWarsaw, Warsaw, Poland

Aniela Brodzikowska

Department of Conservative Dentistry, Medical University of Warsaw, Warsaw, Poland

The aim of this study was to examine the effects of ConcanavalinA (Con A) administration on the early (preosteogenic) and latestages of osteogenesis induced by implantation of demineralizedmurine incisors into syngeneic mice. Local administration of ConA resulted in an increased yield of demineralized incisor-inducedbone when injected during the preosteogenic stage of induction.This effect was not observed when Con A was injected afterheterotopic osteogenesis had been established. This suggests thatCon A recruits osteoprogenitor cells, but does not stimulatedifferentiated chondroblasts and osteoblasts.

Keywords Con A, Dentine, Ectopic Osteogenesis, Immunomodula-tion, Mice

INTRODUCTIONHeterotopic (ectopic) bone formation can be induced in

numerous ways. In some species, it is induced by intramuscularimplantation of transitional epithelium (urothelium). Ectopicbone formation can also be induced by implanting epithelialcell lines, such as KB, HeLa, WISH [1, 2], and by implantingdemineralized bone or dentine matrix intramuscularly [3]. Theability of demineralized tooth matrix to induce endochondralbone formation is well known phenomenon and is similarto that observed by demineralized bone matrix [4–8]. Theinductive molecules were characterized and named osteogeninand amelogenin [8, 9–14]. According to Reddi, soluble signals

Received 8 January 2008; Accepted 11 July 2008; Revised 14 May2008.

Address correspondence to Krzysztof Włodarski, MD, PhD, Chairand Department of Histology and Embryology, Center for BiostructureResearch, Medical University of Warsaw, 5 Chalubinskiego Str, 02-004Warsaw, Poland. E-mail: krzysztof.wlodarski@wum.edu.pl

released from demineralized bone and tooth matrices sculpt“osteosomes,” term denoting the smallest quantum unit of bonethat has all the ingredients of bone [15]. The historical progressin the isolation of osteogenin, bone differentiation factor residentin bone, and dentine was reviewed by Reddi et al. [8] and Reddi[16]. The regulation and maintenance mechanisms of ectopicbone formation are not fully understood [17]. These mechanismsmay differ from those of orthotopic bone formation becauseheterotopically induced bone tissue lacks true periosteal andendosteal membranes [18].

Mechanical influences and hormones can initiate remodelingin orthotopic bone. Inflammatory disorders also profoundlyaffect bone homeostasis [19, 20]. During chronic inflamma-tory reactions, such as rheumatoid arthritis, the recruitmentof inflammatory cells, macrophage-lineage cells, and otherimmune response mediators initiate bone remodeling [21]. Forexample, T cell activation triggers production of interleukin-6(IL-6), which is responsible for bone loss in patients withrheumatoid arthritis [22]. Tumor necrosis factor (TNF), acytokine released during inflammation, increases the numberof osteoclast precursors, which results in bone resorption[23, 24]. Recently, B lymphocytes, which were activated bylipopolysaccharide (LPS), were shown to be involved in boneresorption by enhancing osteoclastogenesis [25].

These mediators are also involved in physiological andpathological bone remodeling [19]. For example, interleukin-1(IL-1) plays a crucial role in inflammation-induced periapicalbone destruction and lesion expansion around the tooth [26].Isolated human osteoblast proliferation may be either stimulatedor inhibited by inflammatory cytokines, depending on cytokinestrength and the duration of exposure [27]. In the diaphysealbone marrow of adjuvant-treated rats, epithelioid cell granulo-mas induce endosteal bone formation [28].

1

Con

nect

Tis

sue

Res

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Mic

higa

n U

nive

rsity

on

10/3

1/14

For

pers

onal

use

onl

y.

2 P. K. WŁODARSKI ET AL.

When Corynebacterium LPS is administered locally aroundlimb bones in mice, both periosteal bone formation andresorption increase [29]. LPS amplifies osteocalcin productionby osteoblasts, but the effects vary between different strains ofmice [30].

Endoprosthetic wear results in polyethylene particles, whichare phagocytosed by macrophages. The macrophages expressinflammatory cytokines that inhibit osteogenesis and activateosteoclastogenesis. This results in endoprothetic loosening inpatients with joint arthroplasty [31].

To examine the impact of specific inflammatory products onlocal bone induction, we implanted demineralized syngeneicincisors into murine muscles. Concanavalin A (Con A) wasinjected into the implantation site. Con A is T cell mitogen,which elicits a strong inflammatory reaction when injected intomuscles that includes granulomatous lesions and sometimes cu-taneous necrosis. The granulomas contain epithelial cells, fibrindeposits, giant cells, fibroblasts, and new blood vessels. Con Ais also a potent activator of osteogenesis and chondrogenesis.

The aim of this study was examination of the timingof immunomodulation by Con A on the effectiveness ofendochondral bone induction by demineralized murine lowerincisors and the duration of demineralization procedure of thesetooth on the outcome of induction.

In this study Con A was administered either at an early pre-osteogenic stage of bone induction (4 days post-implantation)or at a late stage (12 days post-implantation) when chondro- andosteogenesis is advanced.

Bone formation induction by demineralized incisors wascompared under different conditions.

MATERIALS AND METHODS

Incisor Matrix PreparationThree to five-month-old adult Balb/c inbred mice of both

sexes were used in our experiments in accordance with theMedical University of Warsaw guidelines for the care and useof laboratory animals. The mandibles of the mice were removedand immersed into 0.6 N HCl, chilled on ice. After 60 min, theincisors were removed from the alveolar bone and transferredto a fresh HCl solution where demineralization continued foranother 3 hr or for 21 hr. Thus the duration of demineralizationwas 4 and 22 hr, respectively. The dental pulp in the crown androot of each incisor was removed by squeezing the tooth. Andthen the incisors were either washed in distilled water and frozenat –24◦C until implantation or were implanted immediatelyfollowing washing. The mean wet weights of the incisors weresimilar for both demineralization periods (approximately 5 mg).

Implantation of Demineralized IncisorsThe animals were anesthetized with an intraperitoneal

injection containing Rompun (Bayer AG, Leverkusen, Holland)and Calypsol (G. Richter, Budapest, Hungary). After shaving,

short (4–5 mm) longitudinal skin and muscle incisions weremade on the inner side of the left and right thighs. The incisormatrix was inserted into the muscle pocket, the skin was suturedwith a 3-0 Dexon “S” polyglycolic acid suture, and the woundswere disinfected with 70% alcohol.

Individual recipient mice received bilateral implants fromone set of prepared incisors. At various intervals after implanta-tion, the left or right limb was injected with Con A (Pharmacia,Sweden) [0.1 mg Con A dissolved in 0.2 ml of phosphatebuffered saline (PBS)]. On average, half of the animals wereinjected in the left limb and half were injected in the right limb,in order to exclude the influence of manual inconsistencies in thesurgical procedure. The contralateral (non-injected) limb servedas the control.

Animals were sacrificed by cervical dislocation four weeksfollowing incisor implantation to assess bone induction yield.

The implants together with the surrounding thigh muscleswere excised and hydrolyzed overnight in 0.1 N NaOH at 64◦C.The demineralized incisors and soft tissues completely dissolvedduring hydrolysis, without affecting the mineralized tissue mass.Thus, the undigested material recovered was solely bone tissue.These undigested deposits were washed in distilled water anddried overnight at 64◦C. The dry mineral was weighed to anaccuracy of ± 0.1 mg.

The femoral bones from both limbs were excised andhydrolyzed as above. The femoral bone dry mass was measured,providing information on the effect of Con A on adjacentfemoral bone.

Spleens were weighed to measure the general immuneresponse strength.

The popliteal lymph nodes from both legs were excised andweighed accurately to ± 0.1 mg. This measurement was usedto assess the local immune response to the implant and to theimmunomodulators applied.

HISTOLOGYThe implant and the surrounding tissues were fixed in Bouin

fixative, decalcified in a saturated solution of EDTA, embeddedin paraffin and sectioned at 8 µm. Sections were stained withhematoxylin-eosin and examined with light microscopy.

The number of animals, their division into groups accordingto type and immunomodulator administration schedule, and thebone induction yields are shown in the Table 1 and in Figure 2.

Statistical AnalysisThe Student’s t test was used to test for significant differences

between the induced bone yields. For data that was not normallydistributed, the nonparametric Wilcoxon signed-rank test andMann-Whitney tests were applied. Differences were consideredsignificant when the p value was less than 0.05. Statisticalanalysis was performed using SAS V.6.12 for Windows software(SAS Institute, USA).

Con

nect

Tis

sue

Res

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Mic

higa

n U

nive

rsity

on

10/3

1/14

For

pers

onal

use

onl

y.

HETEROTOPIC OSTEOGENESIS BY MURINE DI AT LESIONS SITES 3

FIG. 1. (a). Bone (B) induced by an implanted demineralized incisor (DI)in muscles that were not exposed to Con A. Induced tissues are located at theperiphery of the implant. Scale bar represents 50 µm. (b) Osteogenesis on theperiphery (arrow) and inside the chamber of a DI that was exposed to Con Aduring the early stage of osteogenic induction. A notable inflammatory reactionis observable. Scale bar represents 100 µm. (c) Bone (B) and bone marrow(BM) induced by incisor matrix (IM) in a mouse that was administered Con A12 days post-implantation. Scale bar represents 50 µm.

TABLE 1The effect of demineralization duration in 0.6 N HCl on the

osteoinductive potency of implanted murine incisors

Duration ofdemineralization (hr)

Number ofincisors

Yield ofinduction (mg)

4 60 0.31 ± 0.2∗

22 74 0.23 ± 0.2

Yield is represented as the mean ± SD ∗p < 0.001 via the Wilcoxonsigned test.

RESULTSDemineralized incisor matrix, implanted intramuscularly,

induces bone formation to various degrees. In some cases, boneinduction was not detected.

The weight of limb popliteal lymph nodes exposed to Con Aincreased from 1.4 ± 0.5 mg to 3.8 ± 0.8 mg.

Spleen weights were in the normal range (103–146 mg).These data indicate that a local immune response was producedby Con A.

Limb bud swelling was observed for 2 hr following Con Ainjection and lasted for 4–5 days. In a proportion of cases,skin necrosis was noted. The popliteal lymph nodes wereenlarged, and histological inspection of the Con A-inducedlesions confirmed the presence of granuloma and fibrin deposits,as previously reported [16]. The yield of mineralized boneinduced by implanted incisors that were demineralized for4 hr was higher in the sites exposed to Con A during thepreosteoinductive stage of induction (0.43 ± 0.2 mg) than inthe contralateral control (0.3 ± 0.2 mg; p < 0.05). In contrast,this stimulatory effect was not observed when the Con A lesionsdeveloped during the later stage of osteoinduction (0.35 ± 0.19mg), 12 days post-incisor implantation, when cartilage and bonehad already formed (0.29 ± 0.17 mg; p > 0.05).

Similar results were obtained from implanted incisors thatwere demineralized for 22 hr. Lesions induced by Con A duringthe early stage of bone induction increased heterotopic boneformation (0.41 ± 0.29 mg) compared to the contralateral

FIG. 2. The influence of Con A administration on bone induction yieldproduced following implantation of demineralized incisor matrix into the thighmuscles of Balb/c mice. Error bars represent SD.

Con

nect

Tis

sue

Res

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Mic

higa

n U

nive

rsity

on

10/3

1/14

For

pers

onal

use

onl

y.

4 P. K. WŁODARSKI ET AL.

control (0.2 ± 0.15 mg; p < 0.05). Con A lesions that developedpost-bone formation did not increase bone induction (0.29 ± 0.3mg) more than the control (0.24 ± 0.26 mg; p > 0.05). Theseresults are shown in Figure 1(a)–(c).

The bone yield data were pooled separately for implantedincisors that were demineralized for 4 hr or for 22 hr (Table 1).Implanted incisors that were demineralized for 4 hr resulted ina higher yield of induced bone (0.31 ± 0.2 mg) than prolongedhydrolysis for 22 hr (0.23 ± 0.2 mg; p < 0.001). The durationof demineralization had no effect on the implant weight.

DISCUSSIONEctopic bone induction by demineralized tooth matrix is a

well-known phenomenon [4–8, 12, 15, 16], which is triggered bythe release of bone morphogenetic proteins (BMPs), in bovinedentine identified by Nebgen et al. as amelogenin (presentalso in tooth enamel) [13], from the implant during resorptionby phagocytic cells, such as macrophages and osteoclast-likecells [3]. In our model of bone induction by demineralizedmurine incisors the enamel is removed by the demineralizationtreatment, thus the bone-inducing factor in the murine incisorsare non-collagenous protein not extracted by this procedure. Itis an open question whether the inducing factor is a splicedamelogenin gene, but from the vast literature concerning themolecular characteristic of bone-inducing principals in toothwe consider the cartilage/bone-inducing molecules as bonemorphogenetic proteins in the Urist’s meaning. Rat incisordentin contains factors responsible for initiating the osteogenicresponse upon its implantation into muscles by incorporationof sulfate into proteoglycans [11]. Massive addition of Con Aprobably saturates cells receptors, which bind glycoconiugatesto cell membrane and therefore interacts with glycosaminogly-cans associated to proteoglycans. Orthotopic (periosteal) boneinduction is initiated by cells in the periosteal membranes.Periosteal membrane cells are activated by growth factors thatare released during immune responses. For example, they areactivated by Con A [29] and during spontaneous regression ofMSV-induced sarcoma [32–34]. Reddi et al. named osteogenicgrowth factors present in bone as osteogenin [16].

These studies were designed to determine if Con A applica-tion enhance bone formation following intramuscular implanta-tion of demineralized syngeneic incisors in mice. Additionally,we evaluated the influence of the length of HCl hydrolysis onthe osteoinductive potency of demineralized matrix. Prolonginghydrolysis over 4 hr weakens the osteoinductive potency(Table 1) either by extracting bioactive molecules or by theirdenaturation.

We speculate that Con A or cytokines released followingCon A injection activates chondro/osteoprogenitor cells. It ispossible that Con A injected into the site of tooth matrix bindsto bone inductive molecules present in the implant, as Parlakeret al. reported, thus enhancing the concentration of osteogeninand promoting osteogenesis [35]. The second possibility for

enhancement of bone induction by Con A, seemed to usmore probable one, is the switch from resting chondrocytesinto hypertrophic and calcifying chondrocytes. Con A inducedactivation of resting chondrocytes into metabolically activechondrocytes in vitro was reported by Yan et al. [36].

Con A-induced lesions likely increase heterotopic osteoge-nesis by stimulating early stages of chondro/osteoprogenitorinduction. Osteoinducible cells appear prior to chondroblastsand osteoblasts. Con A-induced lesions have no substantialeffect on existing cartilage or bone when lesions developduring late stage osteogenesis. One possible explanation forthe lack of stimulation of heterotopic osteogenesis by late ConA-induced lesions is that ectopic bone lacks a true periostealmembrane [18]. Con A has been shown to increase chondro- andosteogenesis by activation periosteal/perichondreal membranes[29].

Local immunomodulation affects bone formation. Li et al.[37] reported improved osteogenesis by cytokine BMP-9 whenantibodies to CD4 and CD8 were locally administered, andthis effect was reduced by cyclosporine A. Immunomodulationvia locally administered methylprednisolone on intramuscularlyimplanted, tissue-engineered, autogenous rabbit chondrocytesprotect the implants. These implants were composed of auricularchondrocytes, a polyactide-polyglycolide copolymer coat andspecies-specific fibrin, and induced fibroblast infiltration andtrabecular bone formation in rabbits [38].

Inflammatory reactions in the vicinity of skeletal bonesresult in local bone resorption [39, 40]. Following activatedby inflammatory cytokines, osteoclast precursors produceproinflammatory cytokines and chemokines. Thus, osteoclastprecursors can also be considered as immunomodulatory cells[24].

In some instances, local lesions in soft tissues stimulateosteogenesis to occur [29], and this stimulation is mediatedthrough periosteum activation [1, 2, 32, 41, 42].

Bone formation induced by demineralized tooth matrixlacks a functional periosteum. This explains why, in thepresent experimental work, the activation of osteogenesisby tooth matrix alone is minimal or absent. The increasein bone yield by Con A lesions can be attributed to therecruitment or proliferation of precursor cells, but not to thestimulation of osteocompetent cells, such as chondroblastsand osteoblasts. These lesions, however, have no deleteriouseffect on existing induced bone and do not appear to activateosteoclastogenesis.

ACKNOWLEDGMENTSThis work was supported by a modest grant from the Medical

University of Warsaw (1M15/W2/06). We thank Dr. Wynn Parryand Mrs. Brenda Wlodarski from the University of Liverpoolfor critiquing the manuscript and Mrs. Ewa Wisniewska forlaboratory work.

Con

nect

Tis

sue

Res

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Mic

higa

n U

nive

rsity

on

10/3

1/14

For

pers

onal

use

onl

y.

HETEROTOPIC OSTEOGENESIS BY MURINE DI AT LESIONS SITES 5

Declaration of Interest: The authors report no conflicts ofinterest. The authors alone are responsible for the content andwriting of the paper.

REFERENCES1. Włodarski, K. (1981). Induction of heterotopic and orthotopic cartilage

and bone formation in mice. Acta Biol. Acad. Sci. Hung. 35, 205–218.

2. Włodarski, K. (1985). Orthotopic and heterotopic chondrogenesis andosteogenesis mediated by neoplastic cells. Clin. Orthop. Rel. Res. 200,248–265.

3. Harakas, N. K. (1984). Demineralized bone-matrix induced osteogenesis.Clin. Orthop. Relat. Res. 188, 239–251.

4. Bang, G., and Urist, M. R. (1967). Bone induction in excavation chambersin matrix of decalcified dentin. Arch. Surg. 94, 781–784.

5. Huggins C. B., and Urist M. R. (1970). Dentin matrix transplantation: Rapidinduction of alkaline phosphatase and cartilage. Science. 167, 896–898.

6. Urist, M. R. (1971). Bone histogenesis and morphogenesis in im-plants of demineralised enamel and dentin. J. Oral Surg. 29, 88–102.

7. Ike, M., and Urist, M. R. (1998). Recycled dentine root matrix for a carrierof recombinant human bone morphogenetic protein. J. Oral. Implantolo.XXIV, 113–132.

8. Reddi, A. H. (2000). Initiation and promotion of endochondral boneformation by bone morphogenetic proteins: Potential implications for aviantibial dyschondroplasia. Poult. Sci. 79, 978–981.

9. Katz, R. W., and Reddi, A. H. (1988). Dissociative extraction and partialpurification of osteogenin, a bone inductive protein, from rat tooth matrixby heparin affinity chromatography. Biochem. Biophys. Res. Comm. 157,1253–1257.

10. Luyten, F. P., Cunningham , N. S., Ma, S., Muthukumaran, N., Hammonds,R. G., Nevins, W. B., Wood, W. I., and Reddi, A. H. (1989). Purificationand partial amino acid sequence of osteogenin, a protein initiating bonedifferentiation. J. Biol. Chem. 264, 13377–13380.

11. Veis, A., Sires, B., and Clohisy, J. (1989). A search for the osteogenic factorin dentin. Rat incisor dentin contains a factor stimulating rat muscle cellsin vitro to incorporate sulfate into an altered proteoglycan. Connect. TissueRes. 23, 137–144.

12. Amar, S., Sires, B., Clohisy, J., and Veis, A. (1991). The isolationand partial characterization of a rat incisor dentin matrix polypep-tide with in vitro chondrogenic activity. J. Biol. Chem. 266, 8609–8618.

13. Nebgen, D. R., Inoue, H., Sabsay, B., Wei, K., Ho, C.S., and Veis, A.(1999). Identification of the chondrogenic-inducing activity from bovinedentin (bCIA) as a low-molecular-mass amelogenin polypeptide. J. Dent.Res. 78, 1484–1494.

14. Reddi, A. H. (1998). Role of morphogenetic proteins in skeletal tissueengineering and regeneration. Nat. Biotechnol. 16, 247–252.

15. Reddi, A. H. (1997). Bone morphogenesis and modeling: Soluble sig-nals sculpt osteosomes in the solid state (Minireview). Cell 89, 159–161.

16. Reddi, A. H., Muthukumaran, N., Ma, S., Carrington, J.L., Luyten,F.P., Parlakar, V.M., and Cunningham, N.S. (1989). Initiation of bonedevelopment by osteogenin and promotion by growth factors. ConnectTissue Res. 20, 303–312.

17. De Vlam, K., Lories, R. J., and Luyten, F. P. (2006). Mechanisms ofpathological new bone formation. Curr. Rheumatol. Rev. 8, 332–337.

18. Włodarski, K., and Reddi, A. H. (1986). Heterotopically induced bone doesnot develop functional periosteal membrane. Arch. Immun. Therap. Exp.(Wrocław) 34, 583–593.

19. Goldring, S. R. (2003). Inflammatory mediators as essential elements inbone remodeling. Calcif. Tissue Int. 73, 97–100.

20. Smith, B. J., Lerner, M. R., Bu, S. Y., Lucas, E.A., Hanas, J. S., Lightfoot,S. A., Postier, R. G., Bronze, M. S. and Brackett, D. J. (2006). Systemicbone loss and induction of coronary vessel disease in rat model of chronicinflammation. Bone. 38, 378–386.

21. Gowen, M., Wood, D. D., and Rusesel, G.G. (1985). Stimulation of theproliferation of human bone cells in vitro by human monocyte productswith Interleukine-1 activity. J. Clin. Invest. 75, 1223–1229.

22. Rifas, L., and Avioli, L. V. (1999). A novel T-cell cytokine stimulatesInterleukin-6 in human osteoblastic cells. J. Bone Miner. Res. 14,1096–1103.

23. Włodarski, K., and Włodarski, P. (2006). Osteoclast precursor cell fusionand regulation of mature osteoclast’s activity (in Polish). Post

↪epy Biologii

Komorki 33, 273–284.24. Boyce, B. F., Schwarz, E. M., and Xing, L. (2006). Osteoclast precursors:

Cytokine-stimulated immunomodulators of inflammatory bone disease.Curr. Opin. Rheumatol. 18, 427–432.

25. Kozuka, Y., Ozaki, Y., Ukai, T., Kaneko, T., and Hara, Y. (2006). B cells playan important role in lipopolysaccharide-induced bone formation. Calcif.Tissue Int. 78, 125–132.

26. Kawashima, N., and Stashenko, P. (1999). Expression of bone-resorptiveand regulatory cytokines in murine periapical inflammation. Arch. OralBiol. 44, 55–66.

27. Frost, A., Jonsson, K. B., Nilsson, O., and Ljunggren, O. (1997). Inflam-matory cytokines regulate proliferation of cultured human osteoblasts. ActaOrthop. Scand. 68, 91–96.

28. Tomoda, K., Kitaoka, M., Iyama, K., and Usuku, G. (1986). Endosteal newbone formation in the long bones of adjuvant treated rats. Path. Res. Pract.181, 331–338.

29. Włodarski, K., and Galus, K. (1992). Osteoblastic and chondroblasticresponse to a variety of locally administered immunomodulators in mice.Folia Biologica (Praha) 38, 284–292.

30. Blanque, R., Cottereaux, C., and Gardner, C.R. (1998). Increases ofosteocalcin after ovariectomy are amplified by LPS injection: Straindifferences in bone remodeling. Gen. Pharmac. 30, 61–56.

31. Sosna, A., Radonsky, T., Pokorny, D., Veigl, D., Horak, Z., and Jahoda,D. (2003). Polyethytlene disease. Acta Chir. Orthop. Traumatol. Cech. 70,6–16.

32. Włodarski, K., Kobus, M., and Łuczak, M. (1979). Orthotopic boneinduction at sites of Moloney murine sarcoma virus inoculation in mice.Nature (London) 281, 386–387.

33. Włodarski, K., and Thyberg, J. (1984). Demonstration of virus particles inMoloney murine sarcoma virus-induced periosteal bone in mice. Virchow’sArch. B (Cell Pathol.) 46, 109–117.

34. Włodarski, P., Sevignani, C., Fernandes, M. J., Calabretta, B., andWlodarski, K.H. (2007). Tumour induced by Moloney sarcona virus causesperiosteal osteogenesis engaging osteopontin, fibronectin, stromelysin-1and tenascin. Neoplasma 54, 173–179.

35. Parlakar, V. M., Nandedkar, A. K., and Reddi, A.H. (1989). Affinity ofosteogenin, an extracellular bone matrix associated protein initiating bonedifferentiation, for concanavalin A. Biochem. Biophys. Res. Commun. 160,419–424.

36. Yan, W., Pan, H., Ishida, H., Nakashimi, K., Suzuki, F., Nishimura, M.,Jikko, A., Oda, R., and Katos, Y. (1997). Effects of Concanavalin A onchondrocyte hypertrophy and matrix calcification. J. Biol. Chem. 272,7833–7840.

37. Li, J. Z., Li, H., Hankins, G. R., Dunford, B., and Helm, G. H. (2005). Localimmunomodulation with CD4 and CD8 antibodies, but not cyclosporine A,improves osteogenesis induced by ADhBMP9 gene therapy. Gene Ther. 12,1235–1241.

38. Haisch, A., Wanjura, F., Radke, C., Leder-Johrens, K., Groger, A., Endres,M., Klaering, S., Loch, A., and Sittinger, M. (2004). Immunomodu-lation of tissue-engineered transplants: In vivo bone generation frommethylprednisolone-stimulated chondrocytes. Eur. Arch. Otorhinolaryngol.261, 216–224.

Con

nect

Tis

sue

Res

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Mic

higa

n U

nive

rsity

on

10/3

1/14

For

pers

onal

use

onl

y.

6 P. K. WŁODARSKI ET AL.

39. Shenoy, S. S., and Dinkar, A. D. (2006). Pyogenic granuloma associatedwith bone loss in an eight year old child: A case report. J. Indian Soc.Pedod. Prev. Dent. 24, 201–203.

40. Bounet, J., Zerath, E., Picaud, N., Lesur, C., Mattio, A., Tordjman,C., Hott, M., and Marie, P. J. (1993). Bone morphometric changes inadjuvant-induced polyarthritic osteopenia in rats: Evidence for an earlybone formation defect. J. Bone Miner. Res. 8, 659–668.

41. Włodarski, K. H., Kukwa, A., and Skar_zynski, H. (1987). La-ryngeal carcinomas stimulate locally formation and resorptionof adjacent bones. Bull Pol. Acad. Sci. Biol. Sci. 35, 105–109.

42. Landry, P. S., Marino, A. A., Sadasivan, K. K., and Albright, J. A. (2000).Effect of soft-tissue trauma on the early periosteal response of bone toinjury. J. Trauma. 48, 479–483.

Con

nect

Tis

sue

Res

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Mic

higa

n U

nive

rsity

on

10/3

1/14

For

pers

onal

use

onl

y.