IBMS/ECTS 2001 - PROGRAM and ABSTRACTS
POSTER PRESENTATIONS
All poster presentations will be displayed throughout
the conference.
The suffix after the abstract number indicates the poster session in which the poster will
be attended
(W - Wednesday, T - Thursday, S - Saturday).
Click on the abstract number to view the poster session.
Bone, Cartilage and Connective Tissue Matrix
THE CUT 2000 – A NEW CEMENTLESS HIP SYSTEM WITH METAPHYSICAL ANCHORAGE
J. Scholz*, J. Osel
Auguste-Victoria Teaching Hospital, Center for Orthopaedic Surgery, Berlin, Germany
The CUT 2000 is a new cementless hip system with a short and curved stem for a metaphysical fixation in the femur. The implant is characterized by its form and the surface called Spongiosa Metal II. This macroporous surface structure with a pore depth of up to 3mm and a pore size of 1-2mm similar to that of the cancellous bone, whereby the mesh spaces form an intercommunicating system, has proven bone ingrowth over more than 18 years in standard hip systems. The deep ingrowth of cancellous bone provides an extraordinary stability of the implant.
The new design of the femoral part of the endoprotheses systems consists in a short and curved stem that is mostly inserted in the metaphysis and gets a lateral internal bone contact at the beginning of the diaphysis. The stem is combined with tapers of different lengths and angles of 0, 10 and 20. For the system, a ceramic-ceramic combination of bowl and cup insert is recommended.
Biomechanical testings showing photo elasticity are proving that the load transfer in this implant is similar to normal bone and is preventing any stress shielding. In cases of very late loosenings the removal of this implant is much easier to handle due to a special instrumentation and the insertion of a new standard stem is easily possible due to the not touched diaphysis.
First clinical results after 4 years in 60 cases are showing an excellent Harris Hip Score with an average of 92 points and no clinical, radiological scintigraphic signs of loosening.
THE ASSESSMENT OF BONE MINERALISATION OF DISTRACTION OSTEOGENESIS IN RABBIT TIBIAL MODEL BY PQCT MEASUREMENT
C. W. Chan1*, K. M. Lee2, K. P. Fung3, K. S. Leung1
1Department of Orthopaedics, Chinese University of Hong Kong, HK
2LeeHysan Clinical Reserach Laboratories, Chinese University of Hong Kong, HK
3Department of Biochemistry, Chinese University of Hong Kong, HK
Distraction osteogenesis is a bone regeneration phenomenon due to slow progressive distraction of osseous tissue. This study is investigated the bone mineralisation of distraction gap during consolidation stage assessed by peripheral quantitative computational tomography (pQCT) machine.
After open osteotomy of tibia of adult New Zealand female white rabbits had been performed, an external fixator was applied. Distraction started at the 5th day after osteotomy with 0.5mm, twice a day and continued for 3 weeks to obtain 18mm lengthening. After distraction is completed, the pQCT machine measured bone mineral density (BMD) in 1mm thickness of scanning frame. The %BMD were calculated as % of BMD of measured region is divided by the average of BMD of proximal fragment and distal fragment.
The %BMD of total distraction gap increased by 30% after cessation of distraction for 14 days. However, it became level off for further 14 days. In X-ray radiography, the high intensity in the middle regions of distraction regions was found. It showed that mineralisation took place in different regions of distraction gap. For comparing %BMD, the distraction gap was divided into proximal-gap, mid-gap and distal-gap with equal length. The increase in %BMD of mid-gap contributed mainly on that of total distraction gap (50% increase for first 14 days) From the pQCT tomogram, the change of pixel density concentrated inside the cross-sectional area of distracted callus. It is reflected that the mineralisation of bone occurred inside the distraction callus. The mineralisation was disorganized and took place whole area nearly for the first 14 days. Then distraction callus remodeled for further 14 days. The intramedullary canal became visible and the cortical bone is formed.
The percentage of BMD of distraction gap increased for first two weeks due to mineralisation. Bone mineralisation occurred at the intramedullary region of distraction gap. The increase in BMD is contributed by mainly in mid-gap. The BMD then is steady for further14 days. It is suggested that consolidation phase of distraction osteogenesis consisted of two phases: bone mineralisation initially and bone remodeling phase finally.
COMPARISON OF QUANTITATIVE ULTRASOUND PARAMETERS OF THE OS CALCIS BETWEEN THE RIGHT AND THE LEFT FOOT IN CHILDREN AND ADOLESCENTS
K. Wuensche1*, B. Wuensche2, H. Faehnrich1, S. Vogt1, W. A. Kaiser1
1Institute of Radiology, University Jena, Germany
2Clinic of Orthopaedics, University Jena, Germany
Aim: Exist differences of values of Broadband Ultrasound Attenuation (BUA) und Speed of Sound (SOS) in healthy children and adolescents between the right and the left heel? Which side should be used for measurement?
Method: BUA and SOS were measured in 3299 healthy children and teenagers (1623 girls and 1676 boys), age range 6 - 18 years. Both heels were measured using the Bone Sonometer "SAHARA" (Hologic). Children with diseases influencing bone metabolism were excluded.
Results: There are no significant differences between the right and the left heel for BUA and SOS in all age groups and both sexes (Wilcoxon-test). Further do not exist any association between the values of both sides to left-handed or right-handed persons. The mean of the difference of both heels is for BUA 0.08 (min.-39 and max.38) and is for SOS 1.2 (min.-61.2 and max.58.0).
Conclusion: There is no prefered side for QUS measurement on heel. However right and left side could be slightly different in any person. Thats why always the same side should be used in longitudinal measurements.
EXIST SEX-RELATED DIFFERENCES OF BUA AND SOS OF THE OS CALCIS IN CHILDREN AND ADOLESCENTS?
B. Wuensche1*, K. Wuensche2, H. Faehnrich2, R. Venbrocks1, W. A. Kaiser2
1Clinic of Orthopaedics, University Jena, Germany
2Institute of Radiology, University Jena, Germany
Objective: Aim of the investigation was to detect sex related differences of Broadband Ultrasound Attenuation (BUA) and Speed of Sound (SOS) in healthy children and adolescents in the age range from 6 - 18 years.
Method: For measurement the bone densitometer "SAHARA" from Hologic was used. BUA and SOS were measured in 3299 healthy children and teenagers (1623 girls and 1676 boys), age range 6 - 18 years. Children with diseases influencing bone metabolism were excluded.
Results: SOS was nearly constant during ageing for both sexes. SOS values were significantly higher in 7 years and 13, 14, 15, 16 and 17 years old girls than in boys. BUA values were significantly higher in 9 and 11 years old boys than in girls. Between the age groups 13, 14, 15, 16 and 17 years BUA was significantly higher in girls than in boys.
Conclusion: The above mentioned differences are probably caused by the different onset of puberty, the different onset of growth phases and the effects of estrogen or related factors on muscle-bone relationship. This corresponds to the studies of Rubin et al. [1993] and Ferretti et al. [1998]. Quantitative ultrasound bone densitometry is sensitive to detect small changes of bone mineral density and it is a radiation free measurement to investigate the bone metabolism in childhood and adolescence.
EFFECTS OF HIGH CHOLESTEROL FEEDING ON ARTERIAL AND VESICLE CALCIFICATION USING RABBIT AS A MODEL
H. H. T. Hsu1*, N. P. Camacho2, O. Tawfik1, F. Sun1
1University of Kansas Med. Ctr. Kansas City, KS, USA
2Hospital for Special Surgery, New York, NY, USA
Recent studies indicate that there is a positive correlation between the extent of aortic calcification and osteoporosis. The factors responsible for these paradoxical observations remain unknown. Atherosclerotic calcification may weaken the aorta wall and thereby lead to rupture of the vessel. The mechanisms whereby aortas undergo calcification remain unclear despite numerous studies. Evidence derived from ultrastructural and in vitro calcification experiments suggests the key role of membrane vesicles in atherosclerotic calcification. To further support this role of vesicles, the calcification patterns of aortas and isolated vesicles at early and later stages of atherosclerosis were evaluated using an animal model. To induce atherosclerotic calcification, rabbits were exposed to a 1% cholesterol-enriched diet. After 3 months of the dietary intervention, atherosclerotic lesions were fully developed. Fatty streaks were predominant in the areas proximal to the heart and became less frequent in the distal areas. However, calcification at this stage was not identifiable histochemically or using Fourier transform spectroscopy (FT-IR). After 6 months of high cholesterol treatment, aortas were partially calcified. Histochemical staining for mineral revealed that calcification appeared to occur mainly in the intimal areas immediately adjacent to the media. For the first time, using Fourier transform microspectroscopy in situ, we have demonstrated that mineral was deposited in rabbit atherosclerotic aortas as an apatite-like phase. To determine whether membrane vesicles play a role in mineral formation in aortas, vesicles were isolated from calcified aortas and then compared their calcifiability at early and advanced stages. An increase in calcifiability of membrane vesicles isolated from rabbit aortas proceeded arterial calcification after 3 months of cholesterol interventions. After 6 months of high cholesterol feeding, calcifiability of vesicles was further increased. Like mineral development in diseased aortas, amorphous calcium phosphate and apatite phases were deposited by vesicles in precalcified and calcified aortas, respectively. Altogether, these data reveal that high cholesterol feeding can lead to arterial calcification and that vesicles may play a pivotal role in dystrophic calcification.
ATREMATE BRACHIOPODS: A SERENDIPITOUS MODEL FOR BONE MINERAL STUDIES OR AN EVOLUTIONARY BIOLOGICAL CURIOSITY?
J. P. Cassella1*, D. I. Walton2, N. Garrington3
1Division of Biological Sciences, Derby University, Derby, UK
2Division of Earth Sciences, Derby University, Derby, UK
3Department of Chemistry, DeMontfort University, Leicester, UK
Brachiopods are marine animals whose soft parts are enclosed within a two-valved shell. Whereas the majority of the shells of the phylum are calcitic, atremate brachiopods form shells primarily of calcium phosphate [Vinogradov, 1953].
Brachiopod shell mineral is identical with francolite, a carbonate containing calcium F- apatite, differing principally in the CO32- contents [Le Geros et al, 1985].
Hydroxyproline found in the intercrystalline shell protein suggests an analogy with bone collagen, although the glycine level is reported as too low to allow triple helix formation [Jope, 1977]. However, fibrillar material, identified as collagen with a periodic banding of 45nm has been observed within the internal surface of the valve of Lingula [Williams et al, 1994].
This study has further examined the nature of the mineral in brachiopod shells using; scanning electron microscopy with energy dispersive X-ray microanalysis (SEM-EDS), Fourier Transform (FTIR) and infra red spectroscopy (IR), inductively coupled plasma atomic absorption spectrophotometry (ICP) and fluoride ion selective electrode analysis (ISE).
The calcium to phosphorus ratio (Ca/P) from the ICP of the shell-based mineral
(Ca/P = 2.12) and from three distinct geological locations (Ca/P mean value = 2.15) were directly comparable. FTIR data from brachiopod and geological mineral were similar. Both ICP and FTIR therefore, suggest an apatite structure with similar fluoride substitutions. The fluoride ion content was determined using ISE to be below the detectable limit of the electrode (>0.01ppm). IR spectroscopy confirmed the presence of CO32-, PO43- and OH- and the presence of F- by triply degenerative PO43- vibrations.
The shift away from higher animals in all research, dictates the search for suitable in vitro or in vivo models, which are more acceptable in the light of public concerns. The phosphatic mineral and the presence of a hydroxproline-containing shell protein, furnish brachiopods with the potential for becoming a useful in vitro model for bone formation studies of the future.
LeGeros RZ, Pan C, Suga S, Watabe N. Crystallo chemical properties of apatite in atremate brachiopod shells. Calcif Tissue Int 37:98-100 1985
Jope M. Brachiopod shell proteins: their functions and taxonomic significance. Amer Zool 17: 133-140 1977.
Williams A, Cusack M, Mackay S. Collagenous chitinophosphatic shell of the brachiopod Lingula. Phil Trans R Soc London B 346:223-266 1994
Vinogradov AP. The elementary composition of marine organisms, pp646-660. Sears Foundation Marine Res., Yale Univ
EFFECT OF DEPHOSPHORYLATION OF DENTIN PHOSPHOPHORYN ON APATITE INDUCTION
T. Saito1*, K. Matsuda1, Y. Miyamoto2,3, T. Uemura2,3, M. A. Crenshaw4
1Health Sciences, Univ. of Hokkaido, Tobetsu, Hokkaido, Japan
2NAIR
3JST
4Univ.of North Carolina
Acidic macromolecles including phosphoglycoproteins, sulfated carbohydrates, acidic proteins, and acidic phospholipids have been associated with biomineralization in vivo. We have been focusing on dentin phosphophoryn (DPP) which has been considered to have a primary role in nucleation of mineral in dentin. We found that apatite induction time increases with progressive dephosphorylation of DPP, and the minimum amount of phosphate esters requires for apatite induction. The objective of this study was to clarify the effect of phosphate esters of DPP on hydroxyapatite nucleation by DPP using Nielsen's nucleation theory.
DPP was covalently cross-linked to the collagen fibrils with divinylsulfone. DPP crosslinked to collagen fibrils was partially dephosphorylated with potato acid phosphatase. The amount of remaining organic phosphate ester was measured in dephosphorylated DPP. Samples were incubated at 37 deg C in metastable solutions that do not occur spontaneous precipitation, and were taken at several points. Samples were analyzed for calcium by atomic absorption, and mineral induction time was determined. Samples were analyzed using a NanoscopeIII AFM equipped with a Multimode head and a D scanner, and also observed with an SEM. Interfacial tension was calculated described by classical nucleation theory in each sample.
The phosphate analysis showed that 53.4±7.1 mmole DPP/ mole collagen crosslinked to collagen fibrils. Intact and three degrees (approx. 50%, 70%, and 95%) of dephosphorylated DPP were obtained. The intact DPP, 50%- and 70%-dephosphorylated DPP induced mineral formation in the mineralizing solution, and mineral induction time increased with progressive dephosphorylation of immobilized DPP. We confirmed by X-ray diffraction that the mineral induced was hydroxyapatite. Ca/P molar ratio was determined to be 1.58. On the other hand, 95%-dephosphorylated DPP did not induce mineral formation. The interfacial tension for each of these preparations was calculated from the slope of a plot of log(induction time) vs. (log saturation)-2. Interfacial tension for hydroxyapatite nucleation on immobilized DPP increased with progressive dephosphorylation.
These data indicate that mineral induction activity of DPP is dependent on degree of phosphorylation of DPP in vitro.
INVOLVEMENT OF FIBROBLASTIC CELLS SYNTHESIZING BONE MORPHOGENETIC PROTEIN (BMP) -2 AND BMP RECEPTOR IN CALCIFIED TENDINITIS OF ROTATOR CUFF TENDONS
T. Nakase*, K. Sugamoto, A. Myoui, T. Tomita, T. Miyaji, M. Horiki, N. Tamai, K. Kuriyama, J. Hashimoto, H.Yoshikawa
Dept. of Orthop. Surg., Osaka University Medical School, Suita, Osaka, Japan
[Purpose] Calcified tendinitis of the shoulder joint is a common painful condition characterized by ectopic calcification on the rotator cuff tendons. We have recently reported that fibroblastic cells appear close to the calcium deposition, suggesting that this type of cells contribute to calcification process. However, molecules involved in this condition have not yet been fully elucidated. The purpose of this study is to examine the involvement of bone morphogenetic protein-2 signaling in the calcification process of this condition.
[Materials and Methods] Immunohistochemical and RNA in situ hybridization (RNA ISH) analysis of human BMP-2 were performed in five cases of human surgical samples. RNA in situ hybridization analysis of BMP receptor type IB (BMPRIB) was also performed. In order to characterize the cells at the site of calcium deposition, RNA ISH for alpha 1 chain of human collagen type I (Col I) and staining for alkaline phosphatase (ALP) were done.
[Results] The fibroblastic cells lining close to the calcium deposits were positive for BMP-2 protein and mRNA. BMPRIB mRNA was also localized in these fibroblastic cells. These types of cells were positive for Col I mRNA, but were negative for ALP.
[Discussion] The present findings indicate that BMP-2 and BMPRIB signaling was involved at the site of calcium deposition in calcified tendinitis in the rotator cuff tendons. Fibroblastic cells without osteoblast phenotype synthesized BMP-2 and BMPRIB. BMP-2 has been reported to be activated in the calcification process of arteriosclerosis. Similar mechanisms may be involved in calcification process of calcified tendinitis, and BMP-2 signaling may be one of the key factor to stimulate and/or promote ectopic calcification.
[Conclusion] Non-osteoblastic fibroblastic cells appear at the site of ectopic calcium deposition in rotator cuff tendon tissues contribute to calcification process via synthesis of BMP-2 and BMPRIB.
Fig: Localization of BMP-2 mRNA (arrows) close to the calcified area. (Ca: calcium deposition)
CHARACTERIZATION OF SPECIFIC ANTIBODIES FOR FISH OSTEOCALCIN AND ITS USEFULNESS TO INVESTIGATE OSTEOCALCIN TISSUE DISTRIBUTION IN LOWER VERTEBRATES
D. C. Simes*, J. P. Pinto, P. J. Gavaia, M. L. Cancela
CCMAR, Universidade do Algarve, Campus de Gambelas, 8000 Faro, Portugal
Osteocalcin (BGP or Bone Gla protein) is a small acidic protein with 46-50 residues (pI»4.0) that belongs to the family of the vitamin K dependent, Gla containing proteins. This protein is the most abundant non-collagenous bone protein in mammals and has been isolated only from bone and dentine suggesting that it may be expressed only in hydroxyapatite-containing bone tissue. Previous studies suggest that in mammals BGP is an ossification regulator, but its mode of action at the molecular level, in particular in non-mammalian organisms, remains unclear. Although we and others have identified BGP in lower vertebrates (fish and amphibian), no studies have been carried towards further analyzing BGP localization in these organisms. The purpose of the present study was to determine the tissue distribution of BGP in mineralized tissues from a teleost fish, corvina (Argyrossomus regius). BGP purified from corvina was used to develop a specific antiserum which was tested for sensibility and specificity. The results demonstrate that our antiserum can also recognize BGP purified from other teleost fishes including seabream (Sparus aurata), sole (Solea senegalensis) and toad fish (Halobatrachus didactylus). Comparison of the BGP amino acid sequences from all these fishes, as deduced from the corresponding cDNAs, shows that they are highly homologous and constitute a specific subgroup, separated from all other known BGPs.
DETECTION AND LOCALIZATION OF OSTEOCALCIN (BGP OR BONE GLA PROTEIN) IN TELEOSTS BY IN SITU HYBRIDATION AND IMMUNOHISTOCHEMISTRY
P. J. Gavaia1*, C. A. Viegas1, J. P. Pinto1, M. C. Sarasquete2, M. L. Cancela1
1CCMar, Universidade do Algarve, Campus de Gambelas, 8000-810 Faro, Portugal
2Instituto de Ciencias Marinas de Andalucía (CSIC), Polígono Río San Pedro, 11510, Puerto Real, Cádiz, Spain
Although the presence of osteocalcin (BGP) in fish has been known for a number of years, little knowledge exists on the regulation of expression and tissue localization of this protein in lower vertebrate organisms. In this study we have investigated the site of BGP mRNA expression by in situ hybridation using radiolabeled riboprobes and the tissue localization of the mature protein by immunohistochemistry in the senegal sole (Solea senegalensis), zebrafish (Danio rerio) and gilthead sea bream (Sparus aurata) using fish BGP polyclonal antibodies. Larval, juvenile and adult animals were used in order to observe differences in expression throughout development. The in situ hybridation experiments revealed the presence of BGP mRNA in most of the hard tissues such as bone and the calcified cartilage of branchial arches. The presence of BGP mRNA in the studied individuals was confirmed by Northern hybridation of total RNA obtained from specimens of the same age and developmental stage. The immunolocalization of BGP demonstrated that the protein concentrates in the matrix in calcified and calcifying structures, confirming the in situ results. Some signal was also observed in the kidney, which could be due to the presence of a related protein, nephrocalcin, but these results will require further investigation.
EXPRESSION OF CELL DEATH AND CALCIUM REGULATORY GENE PRODUCTS DURING ENDOCHONDRAL BONE FORMATION
F. Y. Lee*, M. Ho, T. Jayaraman, H. M. Dick
Columbia University, New York, USA
Endochondral ossification process consists of orderly differentiation of chondrocytes, mineralization and formation of bone. Chondrocytes are programmed to undergo proliferation, hypertrophy and cell death. Hypertrophy of chondrocytes, increased levels of intra- and extracellular calcium, mineralization of the matrix and cell death are observed in the hypertrophic and mineralization zone of the growth plate.
The purpose of our study is to find out whether mammalian cell death pathways and calcium regulatory protein are involved in the terminal differentiation pathways during endochondral ossification.
Growth plate from mouse and human were prepared for Western blot and immunohistochemical localization after paraffin embedding. Prepared specimens were probed with antibodies raised against calcineurin, phosphorylated Bad, dephosphorylated Bad, Bcl-2 and caspase 9. Western blot confirmed the presence of the above gene regulatory products. Confocal microscopic examination revealed expression of regulatory gene products in the late proliferating and hypertrophic chondrocytes.
Our findings suggest that gradual increase of calcium level in the hypertrophic zone, calcineurin and apoptotic pathways are well coordinated in the growth plate to promote terminal differentiation of chondrocytes, mineralization and bone formation. Further studies will be necessary to unravel the underlying molecular pathways during terminal differentiation of chondrocytes and bone formation.
A NOVEL SPONTANEOUSLY DIFFERENTIATING CHONDROBLASTIC TISSUE CULTURE - A MODEL FOR IN VITRO ENDOCHONDRAL OSSIFICATION SYSTEM
G. Maor
Faculty of Anatomy, Technioin, Haifa, Israel
Primary cartilage-derived cell cultures tend to undergo dedifferentiation: acquire fibroblastic features and loose most characterizations of mature chondrocytes. This is mainly due to the disruption of the close matrix/cell interrelationship typical for cartilage tissue, which is crucial for maintaining the cartilaginous features. Various models for cartilage tissue cultures have been suggested throughout the years to cope with this problem. Most of these models are based on creating an artificial environment mimicking matrix-cell close interactions, like spot culture, cultures in soft agar or culturing on various supporting substrata.
In the present study we present an easy and reliable model for spontaneously differentiating chondrocytic culture. Mandibular condyles excised from 3 days-old mice, thoroughly cleaned of all soft tissue, are digested for 3x45 min with 0.1% collagenase. Cells from the last two digestions (5x105 cells/ml) are cultured in DMEM supplemented with100µg/ml ascorbic acid 1mM calcium chloride, 10mM beta-glycrophosphate, 10% FCS and antibiotics. Development and growth of the cultures were estimated by following morphological changes as well as functional. Proliferation was studied by BrdU incorporation into DNA and the expression of PCNA. Differentiation was studied by following the expression (at both protein and mRNA levels) of collagen types I, II and X, proteoglycans and osteocalcin, and by Von-Kossa staining for calcification.
During the first 24-48 hrs cells proliferate intensively, while depicting fibroblast-like (long spindle shaped) morphology, and producing mainly type I collagen. Gradually proliferation rate declines, cells gain a polygonal shape and start to produce mainly type II collagen. In the10-14 days old cultures, cells begin to pile up in cartilage-like nodules exhibiting positive staining for: acidic Alcian Blue, type X collagen and Von- Kossa. These results indicate that condylar-derived cells, obtained from neonatal mice, undergo spontaneous differentiation and exhibit features of mature chondrocytes which typically occupy skeletal growth centers. In conclusion, the present study offers a model for chondrocytic tissue culture which might serve as a model for in vitro endochondral ossification model system.
THE USE OF FLUORESCENCE ACTIVATED CELL SORTING TO ANALYSE MATRIX VESICLES FROM EPIPHYSEAL GROWTH CARTILAGE
I. Schoenmakers1,2*, B. M. Gadella1, P. R. van Weeren2, J. F. H. M. Brouwers1, A. Barneveld2, L. M. G. van Golde1, C. H. A. van de Lest1,2
1Department of Biochemistry, Cell Biology and Histology, Faculty of Veterinary Medicine, Utrecht University, The Netherlands
2Department of Equine Sciences, Faculty of Veterinary Medicine, Utrecht University, The Netherlands
For the understanding of the role of matrix vesicles (MV) in the process of endochondral ossification, analysis of individual particles from different zones of the cartilage, would be ideal. The commonly used isolation techniques do however not allow this detailed analysis. We therefore developed a new and quick method to isolate and analyze MVs with an intact membrane, including the use of fluorescence activated cell sorting (FACS). The use of FACS has many advantages, since multiple parameters of one particle can be studied simultaneously, on the basis of which sub-populations can be defined and sorted. In addition, only small samples are required.
Chondrocytes and MVs were isolated from collagenase digested epiphyseal cartilage of 6 weeks old healthy pigs by low speed centrifugation, after which collagenase and other dissolved proteins were removed from the MV-containing samples by gel filtration. For the validation of this technique, the isolated MVs were analyzed with the aid of transmission electron microscopy and biochemical techniques and were compared with chondrocytes.
FACS analysis revealed non-coagulated particles with an intact membrane, respectively with (chondrocytes) and without (MVs) DNA. MV had high content of phosphatidylserine, sphingomyelin, annexin V and alkaline phosphatase in comparison to chondrocytes, and appeared as round, granulated structures surrounded by a lipid membrane.
On the basis of these results, we are confident that the isolated particles are indeed intact chondrocytes and MVs, respectively.
In conclusion, this method of isolation and analysis allows the study of the physiology of intact chondrocytes and MVs during the process of endochondral ossification, and requires only small samples. This opens the possibility to perform detailed investigations on normal cartilage and cartilage in which endochondral ossification is disturbed.
GENETIC MAPPING OF MAJOR SUSCEPTIBILITY LOCUS FOR OSSIFICATION OF THE POSTERIOR LONGITUDINAL LIGAMENT OF THE SPINE TO CHROMOSOME 21Q BY MEANS OF GENOME SCREENING.
K. Ikari1,2*, K. Furushima1,2, S. Harata1, S. Maeda2, I. Ituro2
1Department of Orthopedic Surgery, Faculty of Medicine, Hirosaki University, Aomori, Japan
2Division of Genetic Diagnosis, Institute of Medical Science, University of Tokyo, Tokyo, Japan
Ossification of the posterior longitudinal ligament of the spine (OPLL) is characterized by ectopic ossification in the spinal ligaments leading to a various degree of myelopathy by a compression of the spinal cord. The incidence of OPLL in the general Japanese population was reported to be 1.9 - 4.3% over 30 years of age. The disease has a substantial genetic component, a risk in siblings compared to general population risk (lambda s) of 10. We have previously provided suggestive evidence of linkage to HLA region at 6p21.3 and a candidate gene, collagen 11A2, was screened for possible causality. A molecular variant in intron 6 (T to A substitution at 4 bases upstream from the start of exon 7) was associated with OPLL in case-control comparison. To define the genetic causalities of OPLL in systematic manner, we performed a genome-wide linkage study at an average resolution of approximately 9 cM (a set of 406 polymorphic microsatellite markers) in 140 sib pairs from 71 Japanese families. Non-parametric linkage analysis was performed with affected sib-pairs by the use of two different programs. Identical by descent (IBD) was tested for possible linkage with SIBPAL from S.A.G.E. package. Multipoint linkage analysis was performed using GENEHUNTER. By screening the whole genome, four principal loci of possible linkage were identified on chromosome 6, 14, 16, and 21. The most supportive evidence of linkage was observed with D21S263 at chromosome 21q (P=0.000009) by SIBPAL. Multipoint linkage analysis revealed that the peak linkage was also located close to D21S263 (NPL score = 3.4, P=0.0003). These results strongly indicate that there is a major susceptible gene or genes of OPLL on chromosome 21q22.1.
ANALYSIS OF GENES REGULATED BY CTGF/HCS24 IN LIGAMENT CELLS FROM PATIENTS WITH OSSIFICATION OF THE POSTERIOR LONGITUDINAL LIGAMENT
K. Akaishi*, K. Furukawa, Y. Yamamoto, K. Ueyama, M. Tannno, S. Motomura, S. Harata
Hirosaki University, Hirosaki City, Japan
Ossification of the posterior longitudinal ligament of the spine (OPLL) is characterized by ectopic bone formation in the spinal ligament, mainly through endochondral ossification. CTGF/Hcs24 plays a major role in endochondral ossification. Here, we studied the expression of CTGF/Hcs24 in ossified ligament tissues derived from OPLL patients, and analyzed the genes regulated by recombinant CTGF/Hcs24 (rCTGF/Hcs24) in cultured ligament cells derived from OPLL patients. Immunohistochemical staining revealed that chondrocytes in the transitional region from nonossified to ossified ligament were stained with an antibody against CTGF/Hcs24. Using differential display RT-PCR (DD-RT-PCR) technique between cultured ligament cells derived from a male patient treated with rCTGF/Hcs24 (30ng/ml) for 24 hours and ones not treated, a down-regulated cDNA fragment by rCTGF/Hcs24 was isolated. The sequence matched previously entered GenBank mRNA for homo sapiens breast-ovarian cancer susceptibility gene (BRCA1). Down-regulation in cultured spinal ligament cells derived from another five male OPLL patients was confirmed by RT-PCR analysis. And in all five OPLL patients, the expression of estrogen receptor-beta mRNA was detected by RT-PCR analysis. Furthermore, to investigate the role of BRCA1 in ectopic bone formation, we compared the expression of ALP mRNA as an osteogenic differential marker in the following conditions. In A group, the ligament cells derived from OPLL patients were treated without 17-alpha estradiol (E2) in the absence of rCTGF/Hcs24. In B group, the cells were treated without E2 in the presence of rCTGF/Hcs24. In C group, cells were treated with E2 in the absence of rCTGF/Hcs24. In D group, cells were treated with E2 in presence of rCTGF/ Hcs24. From the results, the expression of ALP mRNA in D group by RT-PCR analysis was significantly higher than any other group. These results suggest that the interaction of E2 and CTGF/Hcs24 on the cells in OPLL patients may be related to the BRCA1 that has the potential to suppress estrogen-dependent transcriptional pathways.
MOLECULAR EVENTS CAUSED BY CYCLIC STRETCH IN SPINAL LIGAMENT CELLS FROM PATIENTS WITH OSSIFICATION OF THE POSTERIOR LONGITUDINAL LIGAMENTS
M. Tanno1*, K. Furukawa2, K. Ueyama1, S. Harata1, S. Motomura2
1Dept. of Orthopaedic Surgery, Hirosaki University School of Medicine, Hirosaki, Japan
2Dept. of Pharmacology, Hirosaki University School of Medicine, Hirosaki, Japan
Ossification of the posterior longitudinal ligament of the spine (OPLL) is characterized by ectopic bone formation progression in the spinal ligaments. Mechanical stress, which acts on the posterior ligaments or ligamentous enthesis of vertebral bodies, is thought to be an important factor in the progression of OPLL. However, the mechanism by which mechanical stress promotes the ossification of ligaments in OPLL is still unknown. Recently, the spinal ligament cells derived from OPLL patients (OPLL cells) have been shown to have several phenotypes for osteoblast, suggesting the metaplagia of spinal ligament cells to osteoprogenitor cells in OPLL. From these evidences, we hypothesized that in OPLL, mechanical signals may be transmitted to induce spinal ligament cell differentiation to osteoblast or to stimulate the release of some cytokines that participate in the ossification processes in spinal ligaments. To examine these possibilities, we investigated the effects of in vitro cyclic stretch (120% peak to peak, at 1Hz) on cultured spinal ligament cells derived from OPLL and non-OPLL patients. In this study, we found that the mRNA expression of alkaline phosphatase (ALP), osteopontin, bone morphogenetic protein (BMP)-2 and its receptors (BMPR-IB and II) were significantly increased by cyclic stretch in OPLL cells, as compared with the cells maintained under resting state, whereas no change was observed in non-OPLL cells. Furthermore, the synthesis and secretion of active BMP-2 into the conditioned medium of OPLL cells were confirmed by Western blot analysis and the induction of alkaline phosphatase activity in a OPLL cell assay, but not in non-OPLL cells. Gd3+, a specific inhibitor of stretch-activated Ca2+ channels, and specific inhibitors of voltage-dependent L-type Ca2+ channels such as diltiazem and nifedipine suppressed stretch-induced ALP activity to resting state level, represents persuasive evidence for a role of Ca2+ influx in signal transduction of mechanical stress to the osteogenic response of OPLL cells. In summary, this study provides first evidences that mechanical stress plays a key role in the progression of OPLL perhaps through the induction of osteogenic differentiation in spinal ligament cells and promotion of the autocrine/ paracrine mechanism of BMP-2 in this lesion.
STRONTIUM RANELATE INCREASES CARTILAGE MATRIX FORMATION
Y. Henrotin1*, A. Labasse1, P. Galais2, Y. Tsouderos2, J. M. Crielaard1, J. Y. Reginster1
1Bone and Cartilage Metabolism Research Unit, University Hospital, CHU Sart-Tilman, Liège, Belgium
2Institut de Recherches Internationales Servier, Courbevoie, France
Based on previous studies demonstrating that strontium ranelate (S12911) modulates bone resorption in osteoporosis, it could be hypothesized that this drug is also active on cartilage degradation in OA. This was investigated in vitro on normal and osteoarthritic human chondrocytes treated or not by IL-1beta. This model mimicks in vitro the imbalance between chondroformation and chondroresorption processes observed in vivo in OA cartilage. Chondrocytes were isolated from cartilage by enzymatic digestion and cultured for 24 to 72h with 10-4 to 10-3 M strontium ranelate, 10-3 M calcium ranelate, or 2 10-3 M SrCl2 with or without interleukin-1beta (IL-1beta) or insulin-like growth factor-I
(IGF-I). Stromelysin activity and stromelysin quantitation were assayed by spectrofluorimetry and Enzyme Amplified Sensitivity Immunoassay, respectively. Proteoglycans (PG) were quantified using a radioimmunoassay. Newly synthesized glycosaminoglycans were quantified by labelled sulphate (Na235SO4) incorporation. This method allowed the PG size after exclusion chromatography to be determined.
Strontium ranelate, calcium ranelate, and SrCl2 did not modify stromelysin synthesis even in the presence of IL-1beta. Calcium ranelate induced stromelysin activation whereas strontium compounds were ineffective. Strontium ranelate and SrCl2 both strongly stimulated PG production suggesting an ionic effect of strontium independent of the organic moiety. Moreover, 10-3 M strontium ranelate increased the stimulatory effect of IGF-I (10-9 M) on PG synthesis but did not reverse the inhibitory effect of IL-1beta. Strontium ranelate strongly stimulates human cartilage matrix formation in vitro by a direct ionic effect without stimulating the chondroresorption processes. This finding provides a preclinical basis for in vivo testing of strontium ranelate in osteoarthritis.
COMPARATIVE HISTOMORPHOMETRIC AND SEMIQUANTITATIVE ANALYSIS OF THE BONE INGROWTH INTO DIFFERENT IMPLANTS IN GOETTINGER MINIPIGS
A. Richter1*, F. Traub1, M. Kratz1, J. Orth2
1Department of Orthopaedic, Philipps University Marburg, Germany
2Department of Orthopaedic, Stadtkrankenhaus, Worms, Germany
Introduction:
There are many studies that show that titan has a great rate of wear in endoprothesis [Williams 1981]. The wear rates of chrome-cobalt-molybdenum are lower [Park 1992] but there are no studies that give evidence for the successful cement-free implantation. This also depends on the difficult sufficient histological preparation. So the purpose of this study was to develop an animal experiment to get better results on these questions.
Material/Methods:
We implant in 18 Goettinger Minipigs test specimens of pure titan in the proximal femur, test specimens of chrome-cobalt-molybdenum in the proximal and distal femur and test specimens of chrome-cobalt-molybdenum coated with hydroxylapatit in the distal femur. After 4, 8 and 12 weeks the pigs were sacrificed. The histological preparation was performed in the cutting and micro-grinding technique and toluidin blue stain. The bone ingrowth was detected semiquantitatively with the help of an optimas system.
Results:
After 4 weeks the uncoated CrCoMb specimens in the proximal femur show the best ingrowth performance compared with all the other specimens. The uncoated CrCoMb specimen in the distal femur showed poorly ingrowth at every time. The uncoated CrCoMb specimen performed all the time a better ingrowth than the pure titan specimen. After 8 and 12 weeks the coated specimen shows the best ingrowth of all.
Discussion:
In this study we could show, that is possible to get sufficient histological results from chrome-cobald-molybdenum implants. The minimum section thickness of our slices was 5 -100 microns. We could demonstrate in our study that chrome-cobalt-molybdenum achieve comparable results like pure titan.
LACK OF ENDOCHONDRAL OSSIFICATION IN LONG BONES OF GROWING FROGS
T. Mori-ishi*, Y. Shibata, A.Yamaguchi
Department of Oral Pathology, Nagasaki university School of Dentistry, Nagasaki, Japan
Vertebrate skeletons have changed their structure depending on environment. There will be several differences in skeletal structure between land animals and aquatic animals. Buoyancy helped behavior of aquatic animals, but land animals should acquire strong skeletons to behave actively with gravity. In addition, skeletons in land animals acquired an important function as a calcium reservoir. These backgrounds prompted us to explore the morphological differences in skeletons between land animals and aquatic animals. In the present study, we investigated the morphological features of femurs in various frogs including Xenopus laevis, Rana catesbeiana, Rana nigromaculata, Rana rugosa and Hyla japonica. Both undecalcified plastic-embedded sections and decalcified paraffin sections were prepared from tadpoles and growing frogs of Xenopus laevis and from the growing frogs of other species. In Xenopus laevis, cartilage core appeared in femoral region of tadpoles at stage 55-56, and then bone collar was formed at perichondrial region of uncalcified cartilage. At stage 57, bone marrow cavity appeared at a central region of the uncalcified cartilaginous core. Cartilage facing bone marrow was not mineralized, though it was comprised of hypertrophic chondrocytes. At boundary between cartilage and bone marrow, no apparent endochondral ossification was observed. Femurs in growing frogs up to 3 cm in body length also lacked apparent endochondral ossification. Structures resembling to endochondral ossification occurred in larger frogs over 4cm in body length, but they were irregular and scant when compared with endochondral ossification in mammalian long bones. Femurs in growing frogs of other species also showed similar features to those observed in Xenopus laevis. Thus, these morphological features in growing frogs are different from those in mammals, because endochondral ossification appeared at boundary between cartilage and bone marrow just after formation of bone marrow during skeletogenesis of long bones in mammals. Taken together, the present study raised two possibilities that explain why growing frogs lack endochondral ossification;1) epiphyseal cartilage in growing frogs lacks factor(s) that stimulates endochondral ossification, and 2) epiphyseal cartilage in growing frogs contains factor(s) that inhibits endochondral ossification.
TROGLITAZONE TREATMENT INDUCES ADIPOGENESIS BUT DOES NOT AFFECT OSTEOGENESIS IN MICE
L. Tornvig2, J. Justesen1, L. Mosekilde2, E. Falk3, M. Kassem1*
1University Department of Endocrinology and Metabolism, Aarhus Amtssygehus, Aarhus, Denmark
2Department of Cell Biology, Institute of Anatomy, University of Aarhus, Denmark
3Institute of Experimental Clinical Research, Skejby Sygehus
Aging is associated with decreased trabecular bone mass and increased adipocyte formation in bone marrow. As osteoblasts and adipocytes share common precursor cells present in the bone marrow stroma, it has been proposed that an inverse relationship exists between adipocyte and osteoblast differentiation. In order to test this hypothesis, we studied mice treated with troglitazone (n=9) given as a 0.2% of food admixture (2.0 g troglitazone per kg food) for 10 months and control mice (n=9). Troglitazone is a potent stimulator of adipogenesis acting at the nuclear receptor: peroxisome proliferator activated receptor-gamma (PPARg). Histomorphometric analysis of proximal tibia was performed in order to quantitate the amount of trabecular bone volume per total volume (BV/TV %), adipose tissue volume per total volume (AV/TV %) and hematopoietic marrow volume per total volume (HV/TV %) using point-counting technique. Bone size did not differ between the two groups. In troglitazone-treated mice, AV/TV was significantly higher than in control mice (5±2% versus 0.2±0.3% respectively, mean±SD, p<0.001). BV/TV was similar in the 2 groups (17±6% for troglitazone-treated group versus 15±5% for control group). HV/TV was reduced in troglitazone-treated mice compared to control mice (78±7% versus 85±6%, respectively, p<0.05) and the presence of vascular sinusoids was observed to be reduced. Our data demonstrate that adipogenesis and osteogenesis can be regulated independently. In addition to its known effects on adipocyte precursor cells, troglitazone-induced adipogenesis may be related to changes in bone marrow vascularity
GENDER-RELATED DIFFERENCES IN BONE AND MUSCLE DEVELOPMENT EVOLVING AT PUBERTY
J. A. Gasser1*, D. Ellison2
1Novartis Pharma AG, Basel, CH
2University of Bristol, Bristol, UK
The aim of our experiments was to monitor gender-related differences in bone and muscle development evolving at puberty. Four week old wistar rats were ovariectomized or sham operated and changes in bone structure, bone mass and cross-sectional muscle area monitored on a weekly basis by peripheral quantitative computed tomography up to the age of 12 weeks. Tibial length was measured on x-rays every week as was body weight gain. All the data was compared to a group of age-related male rats. The onset of menstrual cycles was monitored daily by vaginal smear tests. Body weight, tibial length, cross-sectional muscle mass and all structural bone parameters were identical in all 3 groups until the onset of menarche at 6 weeks. With the appearance of regular cycles in the intact female rats, longitudinal bone growth slowed down rapidly, as did the increase in body weight, cross-sectional muscle area and periosteal bone apposition. There was no difference in mean cortical thickness between the three groups throughout the study. Ovariectomy carried out before the onset of menarche resulted in partial or even complete reversal of the influence of estrogens, with animals being similar to intact male rats. The onset of estrogen production resulted in a rapid increase in cancellous bone mineral density compared to male and OVX female rats which remained constant over time. No distinction could be made between the three groups concerning the relation of muscle area to the polar moment of resistance. Results suggest that the initiation of regular menstrual cycles results in the development of a distinct skeletal phenotype characterized by reduced bone size and length, diminished muscle growth and the development of a special cancellous bone surplus which may be built up for reproductive purposes (late pregnancy, lactation). Female rats which were ovariectomised before the menarche attained a skeletal phenotype reminescent of age-related male rats. Despite of the distinct advantages of male skeletal architecture which evolve around puberty, the muscle bone relation of 24 to 1 remained constant at all times in both sexes indicating that the female skeleton is not at a disadvantage and well adapted to fulfill its mechanical needs.
MICRODAMAGE DISTRIBUTION AND ALTERED MESSENGER RNA GENE EXPRESSION IN THE PROXIMAL FEMUR
N. Fazzalari1,2*, J. Kuliwaba1,2, D. Findlay2, M. Forwood3
1Institute of Medical and Veterinary Science
2Adelaide University
3University of Queensland
Microdamage (Mdx) has been reported in the proximal femur. Mdx repair depends on targeted remodelling which is distinct from microfracture healing. This study describes the distribution of Mdx in the proximal femur and presents a preliminary analysis of the relationship between in vivo Mdx and mRNA gene expression of proteins involved in the regulation of bone remodelling.
A bone biopsy was taken of the proximal femur from 33 patients with primary osteoarthritis. For 15 cases, total RNA was isolated from half of each sample for semi-quantitative RT-PCR analysis of IL-6 and CTR. The other half of the sample was used for Mdx analysis. Controls taken at 12 autopsies were sampled from the subchondral (SC), the medial (M) compressive region and, medial to the greater trochanter (MGT).
In vivo Mdx was identified in 70 micron sections using the basic fuchsin en bloc staining technique. The following were measured, bone volume fraction (BV/TV [%]), crack length (Cr.Le [microns]), crack density (Cr.Dn [#/mm2]), crack surface density (Cr.S.Dn [microns/mm2]) and the damaged bone volume fraction (DxV/BV [%]).
The sampled sites, of the proximal femurs taken at autopsy, had similar Mdx except in the SC region where Cr.Le was significantly smaller. In the region MGT, group comparisons showed no significant Cr.Dn and Cs.S.Dn difference. For OA, Cr.Le was significantly less (OA 51±17 and Controls 63±13) and DxV/BV was significantly increased (OA 5.6±6 and Controls 1.3±2).
In the OA group, IL-6 mRNA gene expression increased as Cr.Dn and Cr.S.Dn increased (Cr.Dn r=0.52, P<0.05 and Cr.S.Dn r=0.58, P<0.02). On the other hand, CTR mRNA gene expression decreased as the Cr.Le increased (r=-0.53, P<0.04).
The SC, M and MGT are sites of compressive loading and similar Mdx. In OA, decreased Cr.Le and increased DxV/BV, may be indicative of an effective inhibition of crack propagation. The increased IL-6 mRNA gene expression associated with increased Cr.Dn and Cr.S.Dn is consistent with an increased stimulus for bone resorption. The decreased CTR mRNA gene expression as Cr.Le increases is indicative of decreased inhibition to bone resorption and/or a decreased number of osteoclasts.
MECHANISM OF BONE ALLOGRAFT FAILURE
M. H. Zheng*, R. Laird, J. Xu, D. Wood
University of Western Australia, Nedlands, Western Australia
Successful reconstructive surgery with allografts is severely limited by a failure rate of 30 – 40%. Allograft failure is due to nonunion of the graft-host junction. The molecular mechanism by which this occurs is not yet fully elucidated. Using a sheep femoral allograft model, we have investigated the cellular and molecular mechanisms associated with nonunion of bone allografts. Five, from a total of twelve operations, resulted in the development of graft-host nonunion, reflecting a failure rate of 42%. Histological assessment revealed that allograft failure was due to the excessive accumulation of and resorption by, osteoclasts (Ocs) on the surface of the bone allograft. Three distinct layers, lying adjacent to the allograft bone surface, in the nonunion groups, were identified. The outer fibroblastic layer contained abundant fibroblasts and connective tissue. Underlying this layer were synovial-like cells and some multinuclear giant cells. The third layer, opposing the bone surface, consisted of Ocs and round mononuclear cells. Histomorphometric analysis showed that allograft unions, featured a large amount of newly formed bone on the surface, (OS/BS = 47.81%) with a small proportion of eroded surface (ES/BS = 20.59%). The number of osteoclasts associated with the allograft bone surface were few (Oc/B.Pm = 1.7190/mm) and activity (ES/BS = 46.68%) of Ocs with a reduced amount of new bone formation (OS = 6.35%). Both calcitonin receptor and H+ATPase mRNA, characteristically expressed by Ocs, were localised to the multinuclear giant cells, indicating that they were Ocs. Synovial-like cells in the histological layer above the Ocs, expressed gene transcript for the Osteoprotegrin Ligand (OPGL), a membrane bound factor that is critical for the induction of Oc activity and osteoclastogenesis.
In conclusion, these findings suggest that failure of bone allografts is partially due to excessive resorption by host Ocs, accompanied by reduced bone formation. The production of OPGL by synovial-like cells, may be responsible for the recruitment and generation of Ocs.
EFFECTS OF INCADRONATE (YM175) ON TYPE-2 COLLAGEN-INDUCED ARTHRITIS IN DA RATS
T. Iwai*, S. Yamamoto, H. Kanoh, S. Fukushima, Y. Okada, K. Takahashi
Pharmacology Laboratories, Institute for Drug Discovery Research, Yamanouchi Pharmaceutical Co. Ltd., Japan
The effects of amino-bisphosphonates (incadronate and alendronate) and indomethacin on arthritis, pain and bone disorder in Dark Agouti strain rats with type-2 collagen-induced arthritis (CIA) were evaluated. Incadronate (1, 3 and 10 mg/kg), alendronate (1, 3 and 10 mg/kg) and indomethacin (1 mg/kg) were administered orally once a day from week 0 to week 4 after immunization. Paw swelling in the control group increased most extremely on week 3 of this study. Bisphosphonates did not significantly prevent the increased paw swelling observed in the control group. The pain threshold for the limbs decreased significantly in week 3 and week 3.5 after immunization. Incadronate significantly inhibited the decrease in the pain threshold compared to the control group, but alendronate did not. In week 4 after immunization, the bone mineral densities (BMDs) of the hind ankle joint, tibial middle shaft and proximal tibia were measured by dual energy X-ray absorptiometry. These BMDs decreased in the control group compared to those in the normal group. Incadronate and alendronate significantly inhibited the decrease in BMD of not only the proximal tibia, but also the tibial middle shaft compared to the control group. Furthermore, incadronate significantly inhibited the decrease in BMD of the hind ankle joint compared to the control group, but alendronate did not. Moreover, the effects of these bisphosphonates on the tibial middle shaft, which consists mainly of cortical bone, were studied by peripheral quantitative computed tomography. Cortical endoperimeter in the control group was significantly lengthened compared to that in the normal group, while there were no differences in cortical exoperimeters among all the groups. It was suggested that the medullary cavity was dilated by immunization. Bisphosphonates significantly inhibited the increase in endoperimeter compared to the control group. Incadronate and alendronate significantly inhibited the decrease in the stress strain index compared to the control group. Thus, bisphosphonates, especially incadronate, inhibited the decrease in pain threshold as well as the bone disorders without affecting the inflammation in the CIA model. These results suggest that incadronate is effective in preventing bone disorders in rheumatoid arthritis.
EFFECT OF TP508, A SYNTHETIC THROMBIN PEPTIDE, ON CBFA1, VEGF, COLLAGEN TYPE II EXPRESSION DURING FEMORAL FRACTURE HEALING
H. Wang*, J. Convery, C. Fowler, J. T. Ryaby
OrthoLogic
TP508 is a synthetic 23 amino acid peptide, representing a receptor-binding domain of human thrombin. Thrombin receptors are expressed on all cells of the mesenchymal lineage. When clots dissolve, thrombin fragments are released and act on thrombin receptors to initiate healing. The aim of this study was to determine the effects of a single percutaneous injection of TP508 on the expression of Cbfa1 and vascular endothelial growth factor (VEGF), two key regulatory factors in bone healing, and collagen type II during femoral fracture healing in rats. Closed fractures at the femur midshaft were created in Sprague-Dawley rats (4 animals/ treatment group/time point). TP508 was injected percutaneously into the fracture site at a concentration of 1 and 10 microg/100microl 24 hours post-fracture for the two experimental groups. PBS was injected in the control group. The calluses of all animals were harvested on days 4, 7, 9, 11, 14, 17, and 21 after fracture. RNA was extracted from calluses, followed by quantitative RT-PCR. The PCR products were quantified after agarose gel electrophoresis using Kodak 1D image analysis software.
RESULTS: 1) At 1microg/100microl, Cbfa1 expression increased 3-fold at 4 and 21 days. Collagen type II was expressed at 4 days in TP508 treated calluses but not in the controls, and was approximately 5-fold greater than the control at 14 and 17 days. The expression level of VEGF was increased at almost all time points. 2) At 10microg/100microl, Cbfa1 was reduced at 4 days but increased at all other time points. Collagen type II expression was increased 2-3 fold at 14 and 17 days; which was less than the degree of stimulation observed with 1microg/100microl TP508. VEGF expression was increased more than 2 fold at 17 days.
CONCLUSION: TP508 has the ability to increase the expression of Cbfa1, VEGF, and collagen type II, thus indicating its potential to promote fracture healing.
CHANGES IN BONE MINERAL DENSITY DURING PREGNANCY AND LACTATION
M. Kaur*, D. Pearson, S. Cawte, P. Baker, D. Hosking
City Hospital, Nottingham, UK
BACKGROUND: Osteoporosis is a common condition affecting 1-in-3 women after the menopause. Changes in bone mineral density (BMD) during pregnancy and lactation are poorly documented and may have long term health implications.
AIMS:
1) To measure changes in BMD during pregnancy.
2) To measure changes in BMD with breast- and bottle-feeding.
METHODS: 97 women considering pregnancy were recruited from the general public via a poster and letter campaign. These women had a baseline BMD measurement performed on the Hologic QDR 2000. Those who conceived and delivered had a scan shortly post-delivery.
46 women were followed from post-delivery to three months post-partum and had DEXA scans performed at these time points. Most were recruited at pre-conception, although some were recruited in early pregnancy.
Pre-conceptual women who did not conceive in a year were invited to return for a follow-up scan.
RESULTS: 41 pre-conceptual women conceived and delivered a singleton term pregnancy. There was a significant fall (median 3.6%) in BMD during pregnancy at the trochanter (p<0.001).
Of the 46 women followed-up post-delivery, 9 bottle-fed, 9 mixed-fed and 28 breast-fed. In the bottle-feeding group, women gained BMD at the lumbar spine (4.4% p=0.02) and trochanter (3.9% p=0.005). Of the women who mixed-fed, there was no significant difference in BMD at any site. Breast-feeders had losses at all sites: lumbar spine (4.4%), total hip (2.2%), femoral neck (2.4%), inter-trochanteric region (3.3%) (p<0.01) and trochanter (0.1% p<0.05).
30 women who did not conceive in the year returned for a second DEXA scan. There was a significant increase in BMD at the lumbar spine (2%), total hip (0.75%), and inter-trochanteric area (1.03%) (p<0.02) and femoral neck (0.25%) (p<0.05); there was no change at the trochanter.
The women who have delivered are presently being followed-up to one year post-delivery, when they will have another DEXA scan.
CONCLUSIONS: Pregnancy is associated with loss of BMD at the trochanter. Breast-feeding is associated with generalised significant losses in BMD. We need to ascertain whether these changes are reversed with time.
PENTADECAPEPTIDE BPC-157 AND RAT TEMPOROMANDIBULAR JOINT OSTEOARTHROSIS
N. Kokic1*, P. Sikiric2, S. Seiwerth2, N. Stambuk2, D. Perovic2, G. Aralica2, P. Konjevoda2, M. Kolombo1, J. Grgurevic1, M.Staresinic2
1Department of Oral Surgery, School of Dental Medicine, University of Zagreb, Zagreb, Croatia
2Departments of Pharmacology, Pathology, Otolaryngology and Surgery, School of Medicine, University of Zagreb, Zagreb, Croatia
Osteoarthrosis (OA) of the temporomandibular joint (TMJ) in the rat was attenuated by a beneficial effect of a stable gastric pentadecapeptide, shown to heal a non-union fracture and deep thickness skin burns, and to reduce adjuvant arthritis, without special carrier, named BPC 157 (GEPPPGKPADDAGLV, M.W.1419). OA-induction included: (i) unilateral condylar articular surface damage (i.e., partial removal), (ii) horizontal incision of both, the collateral ligament at the lateral border of the disk, and the posterior discal attachment, (iii) consequent aggravation by an instability of articular disc and disruption of its vascularization. BPC 157 10 mg or 10 ng/kg b.w. or an equivolume of saline (5.0 ml/kg b.w. (control)) were given intraperitoneally immediately after the surgery. Assessment was at 6 months after surgery. In all directly injured TMJs of controls, relative to the control contralateral undamaged TMJs (P<0.05 / P<0.025, median values (Md)) vertical cartilage fissures, loss of normal cartilage stratification appeared, and relative to the contralateral undamaged TMJs, chondrocyte clustering (number of clusters) was increased (5.5 (Md, undamaged) vs. 14.5 (Md, damaged) while decreased thickness of articular zone (26.3 (undamaged) vs. 13.5 (damaged). In directly injuried TMJs of pentadecapeptide BPC 157 treated rats, with respect to the control, the overal thickness of cartilage was specifically increased, since increased were thickness of cartilage zone (89.3 µm (µg) vs. 66.9 µm (control)) and articular zone (43.3 µm (µg) vs. 13.5 µm (contol)), while decreased were chondrocyte clustering (3.0 (µg), 6.0 (ng) vs. 14.5 (control), and vertical cartilage fissures absent, normal cartilage stratification preserved. In non-surgically altered TMJs the chondrocyte clustering also decreased in pentadecapeptide BPC 157 treated rats relative to control (2.5 (µg) vs. 5.5 (undamaged control)). The significance of this attenuation was especially supported by structural characteristics of pentadecapeptide BPC 157 shown by a computational investigation. Numerous BPC 157 motifs are clustered in collagens and proteoglycans, essential for the cartilage, bone and tooth development, such as collagen a-1 (I), collagen a-1 (IX), aggrecan, fibromodulin precursor, chondroitin sulfate proteoglycan NG2 precursor and cartilage oligomeric matrix protein precursor. Likewise, a special relation over BMP-6 molecule is postulated.
PENTADECAPEPTIDE BPC-157 INCREASED ACHILE TENDOM HEALING AFTER UNILATERAL TRANSECTION IN THE RAT
M. Staresinic1*, B. Sebecic1, T. Sosa1, S. Jadrijevic1, L. Patrlj1, D. Perovic2, G. Aralica2, S. Seiwerth2, N. Kokic3, P.Sikiric4
1Department of Surgery, Clinical Hospital "Merkur", Zagreb, Croatia
2Departments of Pathology, School of Medicine, University of Zagreb, Zagreb, Croatia
3Department of Oral Surgery, School of Dental Medicine, University of Zagreb, Zagreb, Croatia
4Departments of Pharmacology, School of Medicine, University of Zagreb, Zagreb, Croatia
Achile tendom unilateral transection (ATT) in the rat was attenuated by a beneficial effect of a stable gastric pentadecapeptide, shown to heal a non-union fracture and deep thickness skin burns, and to reduce adjuvant arthritis, without special carrier, named BPC 157 (GEPPPGKPADDAGLV, M.W.1419). Assessed 4,7,10,14 days after surgery, tensiometry and microscopical investigation showed a steady healing improvement in saline treated controls, i.e. failure load (N) (1.71, 5.14, 10.42, 9.97), Yang elasticity modul (2.14, 4.66, 6.46, 6.68), fibroblast number (1303, 1763, 1978, 1873) and collagen proportion per visual fields (0.32, 0.40). BPC 157 10 microg, 10 ng or 10 pg /kg b.w. dissolved in saline were given intraperitoneally immediately after the surgery, and thereafter once daily, last application 24 h before sacrifice. With respect to control values (P<0.05 / P<0.017) in pentadecapeptide BPC 157 rats we noted the increased failure load (13.4 (ng), 13.0 (microg), 14 day), Yang elasticity modul (9.43 (microg), 10 day; 10.51 (microg), 14 day), fibroblast number (2019 (ng), 2023 (microg), 7 day; 2364 (ng), 2245 (microg), 10 day), collagen proportion per visual fields (0.49 (ng), 0.50 (microg), 10 day; 0.54 (ng), 0.52 (microg), 14 day). Therefore, pentadecapeptide BPC 157 may positively influence formation and differentiation (maturation), and strength of tissues during Achile tendom healing after surgical transection. The significance of this advanced healing was especially supported by structural characteristics of pentadecapeptide BPC 157 shown by a computational investigation. Numerous BPC 157 motifs are clustered in collagens and proteoglycans, essential for the healing process, such as collagen a-1 (I), collagen a-1 (IX), aggrecan, fibromodulin precursor, chondroitin sulfate proteoglycan NG2 precursor and cartilage oligomeric matrix protein precursor. Likewise, a special relation over BMP-6 molecule is postulated.
APOPTOSIS WITHIN THE GROWTH PLATE IS A FUNCTION OF AGE AND SPATIAL ORIENTATION
A. M. Oberbauer*, J. Cruickshank, D. I. Grossman, T. R. Famula
University of California, Davis, USA
Linear bone growth depends upon proliferation and maturation of growth plate chondrocytes. A critical component to growth plate function is the elimination of the most terminal hypertrophic chondrocytes. Although this removal is known to occur by apoptosis, the extent and developmental regulation of apoptosis within the entire growth plate have not been demonstrated. To address the question of whether the proportion of apoptotic cells is a function of developmental age and physical location within the growth plate, we evaluated costochondral growth plates from 2, 4, and 7 week old rats for the presence of apoptotic chondrocytes. After collection, growth plates were fixed immediately in ruthenium hexamine trichloride. Cells with distinct apoptotic morphology were observed in toluidine blue stained sections and electron photomicrographs of the growth plates at both the chondro-osseous junction and within the region of actively proliferating chondrocytes. Apoptosis was confirmed by Annexin V labeling of isolated hypertrophic and proliferating chondrocytes. Viable and morphologically apoptotic cells in the proliferating and hypertrophic zones were counted in serial sections of the entire growth plates from two to three 2, 4, and 7 week old rats. Cell counts for the lateral regions were analyzed separately from the central growth plate regions. The overall proportion of apoptotic cells was highest at 4 weeks of age (14.2±0.6% of cells) and declined to 3.3% by 7 weeks (P<0.0001). Although the proportion of apoptotic cells was equivalent between the proliferative and hypertrophic zones at 2 weeks of age (P>0.1), more proliferative cells underwent apoptosis at 4 and 7 weeks. In proliferating cells, entire chondrocyte columns appeared apoptotic. The lateral region of the growth plate also displayed a greater degree of apoptosis than the central region at 2 and 4 weeks, but this difference was abrogated by 7 weeks of age. These data indicate that apoptosis plays a role in modulating immature growth plate function. The greater number of apoptotic cells in the lateral growth plate region during the younger ages also suggests a role for apoptosis in maintaining the spatial configuration of the growth plate.
DO THE OSTEOCYTES IN BONE ATTACH TO THEIR SURROUNDING CANALICULI AND LACUNA?
H. Kamioka*, T. Honjo, T. Takano-Yamamoto
Okayama University Dental School, Japan
It is generally accepted that osteocytes are the mechanosensory cells. It is proposed that flow of interstitial fluid through the lacuno-canalicular porosity is the potent candidate for mechanical strain. However, it is not sure whether there is intact flow of interstitial fluid around the osteocyte because the attachment between the extracellular matrix (ECM) and osteocyte is not well documented. On the other hand, focal adhesion is the assembly of structural linkage between ECM and cell surface. Therefore the objective of this study is to examine the distribution of focal adhesions of osteocyte in bone.
To have the preliminary information of focal adhesions of the osteocyte, we first isolated osteocytes by serial digestion of 16-day-old embryonic chick calvariae with collagenase and EDTA and immuno-fluorescently labeled vinculin to visualize focal adhesions in the osteocyte as well as actin fiber to observe whole figure the cell. Secondary, we observed the three-dimensional distribution of the focal adhesions of the osteocyte in vivo, using both confocal laser scanning (CLS) microscopy and differential interference contrast (DIC) microscopy.
Primary osteocytes in vitro showed variety of focal adhesion distribution, such as an osteocyte without focal adhesion, an osteocyte showing focal adhesion only in the base of processes. So, it was suggested that the osteocyte could spread its processes without having an anchor to the substrate. CLS microscopy revealed osteocytes in vivo faintly expressed focal adhesion site in the base of their processes but not in the length of their processes. Complementation of CLS microscopy with DIC microscopy in the same layer confirmed the osteocytes are surrounded by lacuna and canaliculi. However, when the calvariae was incubated in alpha-MEM containing 10% fetal bovine serum for 30 minutes, focal adhesions of the osteocyte distinctly appeared in the base of processes.
It was concluded that osteocytes in vivo formed less attachment to surrounding canaliculi and lacuna. Our results suggest that there is intact flow of interstitial fluid around osteocyte and support the hypothesis that the fluid flow is a candidate for mechanical strain to the osteocytes. Furthermore, it was proposed that the flow of interstitial fluid surround osteocytes might be regulated by serum.
HISTOMORPHOMETRIC ANALYSIS IN ILIAC BONE OF NORMAL KOREAN INDIVIDUALS
K. B. Lee1*, J. H. Kim1, S. J. Kim1, K. M. Yang2, H. Y. Lee2
1Ajou University School of Medicine, Suwon, Korea
2National Institute of Scientific Investigation, Seoul, Korea
Quantitative bone histomorphometric analysis was done in undecalcified bony sections of iliac crests of 114 normal Korean subjects (73 males and 41 females). Each specimen was harvested at autopsy performing 1-2 days after death. The range of age was 3-74 years and grouped by decades. The following parameters were measured: cortical thickness (Ct.Th), trabecular thickness (Tb.Th), trabecular bone surface (BS/TV), total bone volume (TV), trabecular bone volume (BV/TV), cortical porosity, osteoid surface (OS/BS), eroded surface (ES/BS), number of nodes (N.Nd), number of termini (N.Tm) and star volume.
Results showed that TV and BV/TV were decreased with aging (p<0.05) in both male and female. The BV/TV change in female was different from that of male and rapidly fallen down after 5th decade. Node-terminus ratio (N.Nd/N.Tm) was decreased with aging in both sexes (p<0.05). but sexual difference was not significant. The star volume was increased with aging in both sexes, and more rapidly increased after 7th decade in female. The Ct.Th was unchanged before 6th decade in both sexes, but it was rapidly decreased after that period. Cortical porosity in female was higher than that of male (p<0.05), but it was not related to aging. Other parameters were unrelated to age and sex.
BONE GROWTH IN A NOVEL OSTEOCONDUCTIVE MATERIAL FOR ARTIFICAL BONE REPLACEMENT
J. S. Khurana1*, H. Fordyce2, C. Sidebotham3, R. Cohen3, U. Scherbel4, H. Hoslakar1, P. Schultz1, G. Smith2
1Dept. of Pathology, U. Pennsylvania, Philadelphia, PA, USA
2Dept. Orthopedics, Veterinary Medicine, U. Pennsylvania, Philadelphia, PA, USA
3Implex Inc. Allendale New Jersey, USA
4Dept. Orthopedics, U. Pennsylvania, Philadelphia, PA, USA
Background: Bone defects can be extremely difficult to reconstruct. Allograft has the problems of limited availability, risk of transmissible disease, rejection and poor long-term outcome. We investigated a tantalum biomaterial (Hedrocel) that would provide scaffolding, allow bone in-growth and serve as bone replacement.
Design: Six dogs with bilateral mandibular defects received Hedrocel implants (autograft on the contralateral side). Separately, 12 dogs underwent total hip arthroplasty with Hedrocel acetabular and femoral component. Samples were harvested after 6 months, sectioned, embedded in acrylic and ground to 150 microns. Histology and histomorphometry were performed (using digital analysis system and osteometrics software).
Results: Mandibles: 2 dogs developed infection. The remaining 4 showed osteoid crossing the entire implant. 3 (of 4) had central mineralization. There was more bone caudally, superiorly and on the lingual aspects. All controls had union. Osteoid formation was greater in the hedrocel than controls. Mineralization was equivalent. No marrow elements or cartilage were present in the Hedrocel pores. Average bone area was 40%. Hip study: Marrow elements were seen in up to 50% of the sections. Bone area was 13%. Mineralization was greater at the edges (acetubuli) and symmetric (femora). Cartilage was seen in 2 cases.
Conclusion: We showed that Hedrocel can serve as an artificial bone replacement and integrate with bone. A few surprising and interesting results on the type of bone were seen: such as lack of marrow and cartilage formation in the mandible, along with an asymmetric pattern of bone growth. These changes were not seen in the hip. These may be related to its unique shape, biology, embryological origin (intra-membranous) and biomechanics as compared to the hip.
WEAR DEBRIS MEDIATED OSTEOLYSIS IN A RAT FEMORAL HEAD IMPLANT MODEL: WHEN DOES THIS PROCESS BEGIN
D. Fuchs1,3*, P. Shenzer3, E. Sabo2, D. G. Mendes1, D. Lewinson3
1Center of Implant Surgery Bnai Zion Medical Center, Haifa
2Department of Pathology Carmel Medical Center, Haifa
3Department of Anatomy and Cell Biology, Bruce Rappaport
Introduction: Aseptic loosening is the main cause of implant failure in orthopaedic surgery. Loosening is the result of the biological response to debris from the implant material, and is mediated mainly by cytokines, released from inflammatory cells, that serve as osteotropic factors for recruitment and activation of osteoclasts in the bone-implant interface. Material and methods: Using 17 adult female Wistar rats (300-350 gr) we developed an osteolysis model in which the femoral head was replaced by a 316L stainless steel implant. The rats were divided into 3 groups and were sacrificed 7, 10 and 14 days after surgery. Radiologic assessment was performed. For the histomorphometric investigation,
9 samples of 200.00 µm2 from along the implant/bone interface were examined to determine %-eroded bone surface and TRAP+ osteoclasts of more than 2 nuclei were counted in the eroded bone areas.
Results: No early evidence of implant loosening was observed on X-ray. Histomorphometric analysis at 7, 10 and 14 days showed a mean %-eroded area of 1.36µm2 ±0.17; 2.60µm2 ±0.17 and 5.00µm2 ±0.23 [ P<0.0001 (ANOVA) between all groups ]. Mean numbers of osteoclasts were 3±0.38; 4±0.48 & 11±0.72 respectively, [ P<0.0001 (day 7 vs day 10 ; day 10 vs day 14) ]. The calculated ratio of the %-eroded area per osteoclast was 0.44; 0.66 & 0.45 [ P=0.0006 (day 7 vs day 10 ; day 10 vs day 14) respectively.
Discussion: While the peak number of osteoclasts was found on day 14, osteoclast activity, as measured by the ratio of %-eroded surface/osteoclast, was maximal on the 10th day. These are measures of wear debris mediated osteolysis at the beginning of the process, before any X-ray evidence can be observed.
Conclusion: The adult rat femoral head implant is a successful model for experimental aseptic loosening. Currently we are studing the effect of anti-inflammatory and osteoclast suppressing compounds as osteolysis inhibitors.
THE ALTERNATIVELY SPLICED EIIIA SEGMENT IS PREFERENTIALLY EXPOSED TO ANTIBODY RECOGNITION IN AN OSTEOARTHRITIC SYNOVIAL FLUID FIBRONECTIN FRAGMENT SPECIES
J. H. Peters1,2,3*, S. Carsons4, K. Kalunian2, M. Yoshida1, F. Ko1, S. McDougall1,2, T. J. Hahn1,2
1GRECC, West Los Angeles VA Medical Center, Los Angeles, CA, USA
2UCLA School of Medicine, Los Angeles, CA, USA
3Sacramento VA Medical Center, Mather, CA, USA
4Winthrop University Hospital, Mineola, NY, USA
The alternatively spliced EIIIA segment of fibronectin (FN) triggers proteoglycan release and matrix metalloproteinase (MMP) production by chondrocytes in vitro, and an antibody to a loop structure near the center of the EIIIA segment blocks formation of precartilaginous condensations in embryos in vivo. To elucidate possible roles for this segment in chondrocyte dysfunction associated with arthritis, we used Western blot analysis of one- and two-dimensional electrophoretic gels to characterize soluble EIIIA+ species in samples of synovial fluid (SF) derived from patients with osteoarthritis (OA) or rheumatoid arthritis (RA). Similar results were obtained with samples that had been obtained via needle aspiration or arthroscopic lavage. Despite the presence of predominant ~170 and ~200+ kDa species of FN in both OA and RA samples, several monoclonal antibodies (MAbs) recognizing a chondrogenesis-regulating EIIIA loop structure preferentially recognized the ~170 kDa species in OA samples. This species was also stained by MAbs to the amino (N)-terminus and to the cell binding domain, but not to the carboxy (C)-terminus, suggesting a structure spanning from near the N-terminus through the EIIIA segment. Consistent with this, ~170 kDa species that were recognized by MAbs to the N-terminus or to the EIIIA segment both exhibited weak binding to gelatin and possessed similar electrophoretic mobilities in nonreduced-reduced gels and in isoelectric focusing followed by reduced SDS-PAGE. Thus, SF from patients with OA and RA contains soluble ~170 kDa FN fragment molecules that extend from the N-terminus to include chondrocyte-regulatory sequences of the EIIIA segment. Future studies will define the primary structure and function of the various ~170 kDa EIIIA+ SF FN subspecies, which appear structurally similar to a placental FN fragment that was recently observed to trigger MMP production by synovial cells.
IMMUNOLOCALIZATION OF OSTEOCALCIN IN CALCIFIED MICROSPHERES IN ADULT MOUSE BONE USING FITC LABELLING
S. M. Shahtaheri*, J. E. Aaron
Dept. of Human Biology, University of Leeds, Leeds, UK
The noncollagenous proteins (NCPs) in the bone matrix comprise growth factors and a series of other proteins with a less clear biological action, including osteocalcin. Osteocalcin is a calcium binding macromolecule with a high affinity for hydroxyapatite and an uncertain function in calcification.
In order to better understand the role of this bone specific protein in skeletal metabolism it is essential to determine its precise distribution. Using FITC-conjugated method, the immunofluorescence of osteocalcin was examined in cryosections of adult mouse calvaria, lumbar vertebrae and femora. Anti-mouse purified osteocalcin antibody was distributed widely and heterogenously throughout the mineralised extracellular matrix but was absent from osteoid borders. Fluorescence was particularly strong at the cement lines, at sites of tendinous insertion, and in the region of endochondral ossification below the growth plate. Intracellular distribution was also evident in a proportion of osteoblasts and osteocytes from which it appeared to be transported to the calcification front. At all locations staining was associated with particles approximately 1 micrometer in diameter resembling the calcified microspheres which constitute the inorganic phase of immature and mature bone.
It was concluded that osteocalcin was integral to these microspheres where it functions in the containment of calcium phosphate, influencing its form and biomechanical properties.
INCREASED TYPE III COLLAGEN IN THE MINERALIZED COLLAGEN FRACTION OF TRABECULAR BONE IN OSTEOARTHRITIS
S. J. Brown1,2*, H. A. Eriksen3, M. W. J. Davie1, J. Risteli3, C. A. Sharp1
1Charles Salt Research Centre, Oswestry, UK
2Chester Centre for Stress Research, Chester, UK
3Department of Clinical Chemistry, University of Oulu, Oulu, Finland
The bone matrix in osteoarthritis is considered to differ from normal bone. Few studies have characterised the mineralized collagen matrix. In mineralized bone, type I collagen is crosslinked (XL) at the C-terminal telopeptide (C-telo) by divalent and trivalent XLs. The type III collagen (present in minor amounts) N-telo has only been isolated as a trivalent XL structure (IIINTP). In this study these XL collagen-telopeptides and immunoreactive type III procollagen propeptide (PIIINP)-derived antigens have been measured by specific RIAs in trypsin digests of demineralized bone collagen.
A mineralized collagen fraction (MC) was prepared from trabecular bone cores from the superior (SUP) and inferior (INF) regions, drilled from coronal sections of normal ((N) n=11) and end-stage osteoarthritic ((OA) n=21) femoral heads. Bone was powdered, borohydride reduced and stripped of adhering soft tissue by sequential heat denaturation and trypsin treatment prior to EDTA demineralization. MCs were then fully digested with trypsin. ICTP, IIINTP and PIIINP and hydroxyproline (for total collagen) were measured in MC digests. MC digests were also chromatographed on Sephacryl S-100 and the elution patterns of the antigens determined.
Compared with normals the MC of osteoarthritic bone had increased ICTP:collagen ratio (p=0.0005 and p=0.02); IIINTP:collagen ratio (both p=0.004) and PIIINP:collagen ratio (p=0.0006 and p=0.006) in both SUP and INF regions respectively. Chromatography revealed the presence of a major PIIINP peak which was present in all bones (N-SUP 0.9microg/L; N-INF 1.2microg/L; OA-SUP 22.9microg/L; OA-INF 7.4microg/L) and a minor PIIINP antigenic peak which was present in OA bone in both regions (OA-SUP 10.1microg/L; OA-INF 4.4microg/L), and at a lower level in only the N-INF (0.4microg/L) region.
Increased ICTP, IIINTP and PIIINP levels in the MC of bone in end-stage osteoarthritis reflect increased trivalent XL'ing of type I collagen and increased type III collagen formation and maturation within the mineralized matrix. We speculate that end-stage OA is associated with increased amounts of non-mineralized tissue, which becomes protected by mineralization. This may originate from an increased micro-vascular network and/or increased type III collagen synthesis due to the healing of micro-fractures and callus formation.
EXPRESSION OF HEPARAN SULFATE PROTEOGLYCAN CORE PROTEINS DURING IN VITRO-DIFFERENTIATION OF OSTEOBLAST-LIKE CELLS
H. Hausser*, R. Brenner
Division for Biochemistry of Joint and Connective Tissue Diseases, University of Ulm, Ulm, Germany
Bone metabolism is not only regulated systemically by hormones but also locally by a great variety of growth factors and cytokines. Many of these factors have been shown to bind to heparan sulfate chains, and interaction with heparan sulfate has been demonstrated to modulate the biological activity of these factors by a number of different mechanisms, including the formation of heterotrimeric complexes with growth factor and receptor and the catalysis of encounter of receptor and ligand. In addition to the growth factors themselves and their cognate signal transducing receptors, heparan sulfate proteoglycans therefore contribute to the regulation of cell behaviour. The importance of heparan sulfate proteoglycans for bone development and metabolism becomes apparent if one consideres the skeletal phenotypes of genetic defects of their biosynthesis. However, very little is known about heparan sulfate proteoglycans in bone.
In order to advance our understanding of the contribution of heparan sulfate proteoglycans to the regulation of bone metabolism, we studied the expression of the different heparan sulfate proteoglycan core proteins during in vitro-differentiation of osteoblast-like cells using an RT-PCR approach. There was prominent expression of several members of the syndecan and glypican families of membran-associated heparan sulfate proteoglycans and of the matrix-associated molecules perlecan and agrin. Some core proteins exhibited profound changes in their expression levels during the time course of differentiation. Considering the role of heparan sulfate proteoglycans as components of the growth factor reception system, this raises the possibility that osteoblasts change their responsiveness to specific growth factors during differentiation by altering the expression of heparan sulfate proteoglycans.
EXOPOLYSACCHARIDE HE 800 IS A PERFECT EXTRACELLULAR MATRIX FOR BONE HEALING: BIOLOGICAL USE IN CRITICAL SIZE DEFECT IN RAT CALVARIA
P. Zanchetta1*, J. Guezennec2
1Dental University, 29200, Brest, France
2Ifremer, DRVVP, BMM, BP 70, 29280, Plouzané, France
The aim of this study was to evaluate the biological capacities of a bacterial exopolysaccharide from deep-sea vents, named HE 800, to enhance bone healing. The structure of this particular polysaccharide is based on an original repeated linear tetrasaccharide similar to heparin-chondroitin and hyaluronic acid chains. The polymer was used in the critical size defect model in rat calvaria.
Material and method: 5 and 7-mm diameter holes were done on 30 wistar, 7-8 weeks old, male rats calvaria. Animals were sacrified after 15 days and calvaria prepared to histological examination and X-Ray microprobe evaluation.
Results: Defects treated with HE 800 showed bone healing, with a hole filled with a new formed bone. this new tisue was organised in a cortical and trabecular bone with collagen fibers, new blood vessels, osteoblasts and osteocytes. Control side defect showed the same healing. X-Ray microprobe confirmed that mineral composition was the same between old and new formed bone.
Conclusion: HE 800 has a structure nearly identical to extracellular matrix and presents a hydrophobic character. The characteristics of this polysaccharide create an ideal support for bone healing and more than 96% of defect closure was obtained in treated and non treated side due to an pharmaceutical effect of the polymer as results were compared to those obtained with others polysaccharides.
EXPRESSION OF BMPS IN ADULT HUMAN OSTEOPHYTE TISSUE
S. Zoricic1*, I. Maric1, V. Santic2, D. Bobinac1, S. Vukicevic3
1Department of Anatomy, University of Rijeka, Rijeka, Croatia
2Orthopedic Clinic Lovran, Lovran, Croatia
3Department of Anatomy, University of Zagreb, Zagreb, Croatia
Osteoarthritis is characterized by focal cartilage destruction and marked formation of osteophytes. This study was performed on a series of 25 osteophytes removed from the knee and hip joint during joint replacement surgery. Osteophytes were recognized as a small overhanging lips located usually at the margin of articular surface. Specimens were fixed in 4% PFA, undecalcified embedded in MMA and cut serially at 200 mm intervals on 4 mm thick slices. The mechanism of osteophyte formation was examined histologically and immunohistochemically, utilizing specific antibodies for bone morphogenetic proteins (BMPs). Histological analysis revealed loose fibrous tissue on osteophyte's surface and transitional zone of fibrocartilage beneath it. The zone of hyaline cartilage faced to fibrocartilage and showed typical morphology of articular cartilage such as arrangement of cartilage zones and cartilage matrix staining pattern by toluidine blue and safranine O. Osteophytes hyaline cartilage have not contained tide mark line but it had contained large chondrocyte clusters. On the eroded cartilage surface invaded by blood vessels, a mass of osteoclastic and mononuclear cells were visible as well as numerous deposited osteoblasts and osteocytes embedded in osteoid. Fibrous tissue close to bone trabeculae was observed either on surface of osteophytes or within areas of active bone remodeling in osteophytes. BMPs were immunohistochemically observed in: 1) hypertrophic chondrocytes (BMP-3, and -7), 2) osteoblasts (BMP-2, -3, -5, and -7), 3) osteocytes (BMP-3, -6, and -7), 4) bone matrix (BMP-3, -6, and -7), 4) cartilage matrix (BMP-3), and 5) fibrous tissue matrix (BMP-2, and -3). In a typical osteophyte the arrangement of all zones followed normal cellular events present in endochondral and/or intramembranous bone formation. The pattern of BMPs in osteophytes tissue suggest that members of the BMP family may have a specific role in formation of osteophytes removed from osteoarthritic joints.
BIOMECHANICAL PROPERTIES OF FEMORAL SHAFTS IN INMATURE RATS WITH HYPOBARIA-INDUCED HYPOXEMIA
R. M. Alippi*, M. D. Meta, C. Bozzini, M. I. Olivera, J. L. Ferretti, C. E. Bozzini
Department of Physiology, Faculty of Odontology, University of Buenos Aires, Argentina
It has been established that body weight loss and decreased growth rate occur in animals exposed to actual or simulated high altitude (SHA), effects that have been associated with hypophagia because of depressed appetite. The present study was designed to assess the effects of SHA on the biomechanical properties of the femur in weanling Sprague-Dawley female rats. Two groups of animals were studied. The N group was maintained in room air, whereas the H group was exposed to 506 mb (SHA = 5460 m) in a hypobaric chamber. Body mass, body (nose-rump) length and tail length were 60.0%, 82.5%, and 76.3% of N values in H rats at the end of the exposure period. The isolated left femur was analyzed by peripheral quantitative computerized tomography (pQCT) at the midshaft. Analysis of the tomographic scans indicated that bone mass (cortical area, CSA), volumetric cortical BMD (vCtBMD) and spatial fitness of bone material (CSMI) were negatively and significantly affected by SHA. The isolated right femur was mechanically tested in 3-point bending in an Instron mod. 4442 machine for mechanical properties. Femoral length in H rats was 84.6% of N value. Fracture load (48.1%), yield strength (83.3%), and energy elastic absorption (69.3%) were significantly lower in the H group when compared to the N one. Data indicate that exposure to SHA not only adversely affects body mass and longitudinal skeletal growth in prepubertal rats, but also impairs bone mechanical competence. The relative contribution of hypophagia to these bone effects of SHA has not been elucidated.
THE ANABOLIC EFFECTS OF ZINC SUPPLEMENTATION ON SKELETAL STRENGTH IN GROWING RATS
J. Ovesen1,2*, B. Møller-Madsen1, J. S. Thomsen2, G. Danscher1, L. Mosekilde2
1Department of Neurobiology, Institute of Anatomy, University of Aarhus, Denmark
2Department of Cell Biology, University of Aarhus, Denmark
The aim of the study was to asses the skeletal effects of alimentary zinc depletion and supplementation in growing rats. The study was planned as a dose response study, and three different skeletal sites were investigated by mechanical testing and measurements of bone mass and dimensions. 36 male Wistar rats, 4 weeks old, were divided into three different groups of 12 rats each and had free access to a semisynthetic diet differing in zinc content. Group 1 received a zinc-free diet containing 2.042 mg zinc/kg, group 2 received the basal diet containing 47 mg zinc/kg, and group 3 had a diet containing 60 mg zinc/kg. All animals were sacrified 4 weeks after the start of the experiment and the right femora were removed. The biomechanical and morphological effects were measured at the following three skeletal sites: Femoral diaphysis; femoral neck; and distal femoral metaphysis. Biomechanical testing revealed a significant zinc-induced increase of bone strength at all investigated sites. It also showed that zinc acted on the bone strength in a dose-dependent manner except for the distal metaphysis, where there was no significant difference between the group fed with the normal-zinc diet and the group fed with a hyper-zinc diet. However, at all three skeletal sites there was a significant difference between the group fed with hypo-zinc compared with the other two groups.
We conclude that alimentary zinc supplementation in growing rats induced an increase of bone strength in both the femoral neck and the femoral diaphysis. Zinc also improved the rates of growth in the rats. The weight, length and diameter of the femora were all higher in the rats given zinc supplementation. These results further support the view that zinc has a potent anabolic effect on bone metabolism.
P42 S
Withdrawn
BMU-COUPLING IS REGULATED BY LOCAL PATTERNS OF FLUID FLOW
E. H. Burger1*, T. H. Smit2, J. M. Huyghe3
1ACTA - Vrije Universiteit, Amsterdam, The Netherlands
2Vrije Universiteit Medical Center, Amsterdam, The Netherlands
3Technical University Eindhoven, The Netherlands
Introduction - There is evidence that BMU-coupling is regulated by deformation of the bone matrix [1]. Evidence also accumulates, that mechanosensing by osteocytes is related to extra-cellular fluid flow. The purpose of this study is to relate the theory of extracellular fluid flow to BMU coupling. To that end, we determined the pattern of fluid flow around a tunnelling osteon under axial loading.
Methods - The problem is approached with Biot’s theory of poroelasticity, and calculated with the finite element method. The tunnelling osteon was modelled axisymmetrically as a cylindrical gap with a spherical end. The bone matrix was described as an isotropic material with fully saturated lacuno-canalicular porosity. The load applied to the model was that of a person walking at 4 km/h. The maximum deformation of the bone matrix was 1500 microstrain.
Results - On loading, a typical flow pattern appears around the cutting cone: along the the closing cone, fluid is pressed out of the bone matrix, but at the tip fluid flows into the bone matrix. This is due to a local area of volumetric expansion in front of the cutting cone. Inside the bone matrix, an outflow pattern exists along the cylindrical wall, which damps out at a depth of some 0.1 mm. In front of the cutting cone, however, the inflow at the surface changes to an outflow at a depth of some 10 microm. So, just below the surface of the cutting cone, the fluid flow is close to zero. At unloading, the fluid flow pattern is more or less reversed.
Discussion - The pattern of bone fluid flow around a tunnelling osteon was determined for a walking cycle, using Biot’s theory of poroelasticity and a finite element model. The main finding was that the fluid flow pattern is different near the cutting cone as compared to the closing cone, which suggests that the osteocytes within the bone matrix sense different patterns of fluid flow near sites of osteoclastic and osteoblastic activity. This is compatible with the hypothesis that local patterns of bone fluid flow regulate BMU-coupling.
References - [1] JBMR 2000: 301-307
DENSIFICATION BY INFILLING MARROW SPACE IN RESPONSE TO EXERCISE IN THOROUGHBRED HORSE DISTAL CANNON BONE
A. Boyde1*, C. M. Riggs2, E. C. Firth3
1University College London, London, UK
2Oakey Veterinary Hospital, Oakey, Queensland, Australia
3Massey University, Palmerston North, New Zealand
We examined changes induced by training in equine third metacarpal bones (McIII). Distal parts of McIIIs were obtained from controlled training experiments in which 2 year old Thoroughbreds were subjected to strongly contrasting exercise routines. McIIIs were sliced longitudinally in para-sagittal or central and dorsal- and palmar-oblique medio-lateral planes, embedded in PMMA, the blocks micromilled and carbon coated and the bone mineralisation density studied at the cubic micron scale using quantitative digital backscattered electron imaging. Entire bone slices were analysed using automated scanning of sub-fields. For each sub-field, anatomical region, and whole slice, bone volume fraction (BVF) was calculated with mean and median BSE grey levels: volume fractions of 16 density phases were estimated. The fraction of the organ volume occupied by any form of bone tissue in the distal extremities was increased in the exercised group. Most of this extra bone was deposited within the former marrow space in the central regions of the extremities. The bone was of mixed woven and lamellar nature and, where relatively thick, formed in relation to numerous fine blood vessels which it incorporated to form canals. It was deposited upon prior lamellar bone surfaces without the intervention of prior resorption and without the formation of a hypermineralised cement line. In the immediate subchondral bone zone, the open canal or marrow space was much less in the exercised groups, whilst extensive spaces, representing resorption episodes, were more easily seen in the control group. The more loaded immediately subchondral zones, e.g. the palmar regions in the condyles, had the highest BVF, and a lower mean level of mineralisation, with the histology showing repetitive turnover in small domains. The greater amount of bone formed in response to training exercise was of net lower density, reflecting increased material compliance.
COLLAGEN FIBER ORIENTATION: A CHARACTERISTIC OF STRAIN-MODE-RELATED REGIONAL ADAPTATION IN CORTICAL BONE
J. G. Skedros
Utah Bone and Joint Center, Salt Lake City, Utah, USA
Department of Orthopaedics, University of Utah, Salt Lake City, Utah, USA
Skeletal morphologists often examine the structural and material organization of skeletal tissues as a means to interpret loading history. However, it is unclear if even a simple loading history can be reliably inferred from specific structural and material characteristics. Various limb bones were examined to determine if such characteristics exhibit consistent adaptations to customary strain distributions. Each ‘experimental’ bone was subject to an in vivo history of bending in a consistent direction. Notable adaptations were expected because mechanical properties of cortical bone markedly differ in tension, compression, and shear strain modes. Undecalcified mid-diaphyseal transverse segments from mature bones (n=7 each) were embedded in methacrylate: sheep, deer, and horse calcanei, sheep and horse radii, horse third metacarpals (MCIIIs), and sheep tibiae. The horse MCIIIs and sheep tibiae served as ‘controls’ since they experience comparatively complex loading. In the experimental bones, tension and compression strains of unequal magnitudes prevail on opposite (cranial/caudal) cortices; this disparity is diminished along the neutral axis (medial/lateral cortices). Ultramilled (100±5 micron) specimens were viewed under circularly polarized light. Variations in predominant collagen fiber orientation (CFO) were expressed as relative differences in the amount of transmitted light in the cranial, caudal, medial, and lateral cortices. Additional quantitative analyses included fractional area of secondary bone, secondary osteon population density, mean area of secondary osteons, cortical thickness, cross-sectional second moments of inertia along the major and minor axes, and polar moments of inertia. Regional mineral content (ash%) was determined in adjacent segments. Results showed that only CFO exhibited a consistent relationship with loading history – in experimental bones, compression cortices had a significantly more oblique-to-transverse collagen than tension cortices [p<0.017; range of differences: 15% (sheep radii) to 69% (horse radii)]. As anticipated, there were no significant CFO differences between the cranial-caudal and medial-lateral cortices of sheep tibiae and the cranial-caudal cortices of horse MCIIIs. The consistent regional strain-mode-related material heterogeneity may reflect adaptations for specific biomechanically important features of local strain history. In contrast to adaptations that affect global (i.e., whole bone) stiffness/strength requirements, these adaptations may enhance fatigue resistance and fracture toughness for local loading conditions.
ZINC STAINING OF MATRIX METALLOPROTEINASES AND ENDONUCLEASES IN GROWTH CARTILAGE
S. Gomez
Dept Anatomia Patologica, Faculty of Medicine, University of Cadiz, Spain
A new method for Zinc histochemistry was applied to stain zinc atoms from matrix metalloproteinases and endonucleases to localize their distribution in epiphyseal plate rat cartilage. Though these zinc ions are firmly bound and essentially they are not available, drastic ammonium sulfide exposure rendered them reactive for staining. Matrix metalloproteinases were detected in chondrocytes and in extracellular matrix along the longitudinal septa before matrix calcification. A second localization was found at the resorptive limit of calcified matrix adjacent to the zone of vascular invasion. Zinc of endonucleases involved in apoptosis was stained within the nuclei in the last rows of hypertrophic chondrocytes precisely where chromatin was condensed.
BONE MORPHOGENETIC PROTEIN-2 STIMULATES INORGANIC PHOSPHATE TRANSPORT AND MINERALIZATION IN OSTEOBLAST-LIKE CELLS
A. Suzuki1*, J. Guicheux2, I. Sato1, Y. Miura1, Y. Oiso1, J. P. Bonjour2, J. Caverzasio2
11st Dept. Int. Med., Nagoya University, Nagoya, Japan
2Division of Bone Dieases, University Hospital of Geneva, Geneva, Switzerland
Bone morphogenetic proteins (BMPs) play an important role in the development of bone and cartilage. BMP-2 is produced by osteogenic cells including osteoblasts and stimulates the differentiation of preosteoblasts and the activity of osteogenic cells. Inorganic phosphate (Pi) is an important element for the calcification of the bone matrix. Recent studies in cultured MC3T3-E1 cells suggest a specific role of the Pi transport system Pit-1 in initial events of matrix mineralization. The aim of the present study was to analyse whether BMP-2 regulates the expression and activity of PiT-1 and investigate the possible role of this transporter in the BMP-2-induced matrix mineralization. BMP-2 time- and dose-dependently stimulated Na-dependent Pi transport at day=6 in MC3T3-E1 cells. An effect of BMP-2 on Pi transport was detected after 3 hours. It was maximal after 6 hours and remained expressed at least 24 hours. A maximal response was obtained with 30 ng/ml of BMP-2 (2.2 fold). Kinetic analysis indicated that BMP-2 increased the maximal rate (Vmax) of the transport system but did not affect the apparent affinity for Pi. Pretreatment of the cells with either actinomycin D (2.5 microg/ml) or cycloheximide (5 microM) completely abolished the stimulation of Pi transport induced by BMP-2. Northern blotting analysis showed an increased expression of mRNA encoding Pit-1 after 2 hours BMP-2 exposure. In parallel with the stimulation of Pi transport, BMP-2 enhanced both ALP activity and the formation of mineralized bone nodules in differentiating cells.
In conclusion, the results of this study indicate that BMP-2 stimulates the expression and activity of the Pi transporter Pit-1 in osteoblast-like cells via a RNA and protein synthesis dependent process. This effect is associated with enhanced expression of bone matrix mineralization suggesting a possible role of this Pi transport system in bone matrix calcification.
BMP RESPONSIVENESS IN HUMAN MARROW STROMAL CELLS
D. L. Diefenderfer*, G. Grasso-Knight, P. S. Leboy
University of Pennsylvania, Philadelphia, USA
INTRODUCTION: Cultured bone marrow stromal cells from various species have been shown to possess an inducible osteogenic phenotype. Interestingly, the potency of individual inducers is species-dependent. BMP has a relative potent osteogenic effect on rat and mouse stromal cells yet is usually a poor inducer of osteogenesis in cultured human stromal cells. We have been examining why the BMP effect is poor and variable in human cells.
METHODS: Human stromal cells (HMC) were isolated from marrow aspirated from femora during total hip arthroplasty. The marrow was washed to remove fat and the mononuclear cells concentrated on Ficoll-Paque (Amersham-Pharmacia Biotech). Primary cultures were established at 5 x 105 cells/cm2. Media in primary cultures were initially changed on day 3 and, generally, every second day thereafter. Half of the primary cultures from individual samples were treated with dexamethasone (dex; 10-7M), a potent inducer of osteogenesis in HMC. Just prior to confluence, first passage cultures were established at 104 cells/cm2. All cultures were treated with ascorbate phosphate at 100 microg/ ml. Selected cultures were treated with BMP-2 at 100 ng/ ml. Cultures were harvested at day 6 for alkaline phosphatase (AP) assay and total RNA isolation.
RESULTS: Baseline AP activity in first passage cultures derived from dex-treated primaries was higher than activity in cultures derived from non-dex primaries. In most first passage cultures derived from non-dex primaries, BMP-2 had no significant effect on AP activity; some isolates showed modest inhibition by BMP. In first passage cultures derived from dex-treated primaries, BMP-2 significantly increased AP activity (p=0.01). However, the magnitude of this effect varied widely. Interestingly, BMP-2 induced noggin mRNA in first passage cultures irrespective of primary culture condition and AP level in first passage. We conclude that human marrow stromal cells possess a latent osteogenic response to BMP which can be induced by dex treatment.
DISTRIBUTIONS OF MRNAS FOR BMP-2 AND BMP RECEPTOR IN OSTEOARTHRITIC CARTILAGE
T. Miyaji*, T. Nakase, T. Tomita, M. Kaneko, K. Takahi, A. Myoui, K. Kuriyama, M. Horiki, H. Yoshikawa
Department of Orthopaedic Surgery, Osaka University Medical School, Osaka, Japan
[Purpose] Osteophytes are neoplastic tissues made up of osseous and cartilaginous components with fibrous mesenchymal layers. The tissues often originate from the margin of osteoarthritic joints, especially at the synovio-chondral junctions. The aim of this study is to examine the involvement of BMP-2 signaling in the process of cellular differentiation during osteophyte formation in the human.
[Materials and Methods] In situ hybridization (ISH) utilizing digogigenin-labeled cRNA probes for human bone morphogenetic protein (BMP)-2 and BMP receptor IB, and immunohistochemistry (IH) with a monoclonal antibody against human BMP-2/4. To determine the phenotypes of cells, ISH using cRNA probes for human collagens types I, II and III (Col I, II, III) were also performed. Osteophytes obtained at the surgery, with consent, from 8 specimens from 6 individuals were used in this study.
[Results] BMP-2 mRNA and protein were distributed in mesenchymal cells (positive for Col I and III, negative for Col II) overlying or adjacent to apparent osteophytes, and in chondrocytes (positive for Col II) located in neo-plastic hyaline cartilage. BMP-2 was also localized in chondrocytes in fibrocartilaginous (positive for Col II and Col III) and in mesenchymal cells undergoing intramembranous ossification forming osteophyte. In mesenchymal cells although BMP-2 was synthesized by in mesenchymal cells surrounding the osteophyte, mRNA for BMPRIB were predominantly localized in chondrocytes themselves. Neither BMP-2 mRNA and protein was detected in cells in non-osteophytic cartilage. These results is summarized in a Table.
[Discussion] These results suggest that mesenchaymal cells contribute to initiate and promote formation of osteophyte via synthesis of BMP-2. Co-localization of BMP-2 and BMP receptor type IB in cells in mesenchymal layers in mesenchymal cells located in the periphery of osteophyte suggest that BMP-2 may play roles in osteophyte fromation in osteoarthritic cartilage.
ADENOVIRUS MEDIATED BMP-2 GENE THERAPY ENHANCES BONE FORMATION IN A MURINE METAPHYSEAL BONE DEFECT
H. Uusitalo1*, T-J. Gao1, P. Virolainen2, M. Ahonen1,3, H. Aro2, V-M. Kähäri1,3, E. Vuorio1
1Dept. of Medical Biochemisty, Turku, Finland
2Dept. of Surgery, Turku, Finland
3Centre for Biotechnology, University of Turku and Åbo Akademi University, Turku, Finland
We have investigated the capacity of bone morphogenetic protein (BMP)-2 to enhance bone healing in a metaphyseal bone defect model in the mouse. For this purpose a recombinant adenovirus (RAdBMP-2) harboring the complete coding sequence of the human BMP-2 under the control of cytomegalovirus IE promoter was constructed. RAdBMP-2 viruses were injected into the defect site in the distal metaphysis of the femur immediately after surgery. Control defects were injected with recombinant adenoviruses harboring the LacZ gene. The healing process was followed at 7, 14, 21 and 42 days using histology, peripheral quantitative computed tomography (pQCT), biomechanical testing and molecular biologic analyses. Histologically, a characteristic effect of BMP-2 was enhanced osteogenesis within the medullary cavity and periosteal chondrogenesis adjacent to the defect, particularly during the first week of healing. At two weeks, pQCT analysis revealed increased bone mineral content (BMC) in the defect area injected with RAdBMP-2 when compared with the controls. Similarly, an increasing trend was seen in the bending stiffness of the healing femur at two weeks after RAdBMP-2 injection. Analysis of the chondrogenic and osteogenic activity in the defect area by Northern analyses revealed that the mRNA levels for cartilage and bone components in defects injected with RAdBMP-2 remained essentially unchanged, indicating a balanced increase in chondrogenesis and osteogenesis. The production of human BMP-2 in defect area was demonstrated by a reverse transcription-polymerase chain reaction (RT-PCR) assay. The highest levels of recombinant BMP-2 were seen already at one week of healing. In summary, the data demonstrate the capacity of transient overproduction of BMP-2 to induce both chondrogenesis and osteogenesis. As the defect model was developed in the mouse, it can now be tested for the biological activity of other bone inducing factors both in normal mice and in transgenic mice harboring various type of gene modifications using adenovirus mediated gene transfer.
BONE MORPHOGENETIC PROTEINS (BMPS) AND ITS RECEPTORS (BMPR) ARE EXPRESSED BY BOTH BONE FORMING AND NON BONE FORMING LESIONS
J. S. Khurana1*, S. Ogino1, T. McHale1, M. Feldman1, U. Scherbel2, P. Wirganowicz2, P. Schultz1, B. A. Alman3
1Dept. of Pathology, U. Pennsylvania, Philadelphia, PA, USA
2Dept. Orthopedics, U. Pennsylvania, Philadelphia, PA, USA
3Dept. Orthopedics, Hospital for Sick Children, Toronto, Canada
Background: BMPs can cause ossification in soft-tissues and induce bone formation Primary and secondary lesions of the musculoskeletal system can be bone forming or bone destroying. We studied whether BMP expression is limited to bone forming lesions.
Design: We performed RT-PCR with primers for BMPs, BMPR (ALK 3), Northern blots with probes antisense to BMP 2 as well as immunohistochemistry for BMP 2 and 4 and BMPR (BRK 1) on 5 cases of Desmoid tumor (DT), 5 cases of fibrous dysplasia (FD), and 17 cases of a variety of metastatic tumors with a lytic radiographic pattern.
Results: By RT-PCR, BMP (1, 2, 3, 4 and 6) was expressed by DT and FD. Additionally both lesions also expressed BMPR. Northern blot showed that BMP-2 was expressed at a higher level in FD than in DT. Immunohistochemistry showed that BMP 2 and/or 4 protein was produced by both lesions. Of the 17 cases with lytic patterns on x-rays, 7 cases were completely negative for both BMP 2, 4 and BRK-1 (a BMP receptor). 10 cases were at least focally positive for at least one of the BMPs.
Conclusions: Both bone forming (FD) and non-bone forming (DT and lytic carcinomas) expressed BMPs, and BMPR. The BMPs however may still play a role in bone formation despite its expression in both lesions. There are several other BMPs and BMPRs, that we did not test, and a difference in one of these and their relative or absolute amounts could be significant. Additionally, post translational cleavage and dimerisation or the co-expression of other factors may be important in the induction of bone. Bone formation is a complex process, which may involve multiple factors other than or in addition to BMP.
BONE MORPHOGENETIC PROTEIN EXPRESSION IN DIFFERENTIATING OSTEOBLASTS IS REGULATED BY PARATHYROID HORMONE
S. Martinovic*, V. Kisic, F. Borovecki, S. Vukicevic
Department of Anatomy, School of Medicine, University of Zagreb, Zagreb, Croatia
We have recently shown that MC3T3-E1 cells during normal differentiation process in vitro synthesize only BMP-4 which is necessary and sufficient for osteoblastic differentiation and extracellular matrix deposition. In the same system the function of BMP-4 could be replaced by BMP-7, another BMP family member.
In this study we examined the effect of parathyroid hormone (PTH) addition on regulation of BMP gene expression by osteoblastic MC3T3-E1 cells. Cells were routinely cultured and after reaching confluency, beta-glycerophosphate and ascorbic acid were added. At designated time points (days 0, 3, 7, 12 and 17), parallel cultures were treated with PTH (10-9 M)for 12 and 72 hours. Total RNA was isolated using TRIzol reagent and cDNA was synthesized from 4 microg of total RNA with Superscript II RNase H-Reverse Transcriptase (Gibco BRL), and expression of BMP-2 to BMP-7, alkaline phosphatase, collagen type I, osteocalcin, osteopontin, BMP receptors type I (ALK-3 and 6) and G3PDH was analyzed by RT-PCR.
Compared to control samples, the addition of PTH slightly delayed the maximum expression level of BMP-4 mRNA, reached on day 7. After that point, the BMP-4 gene expression gradually decreased in time. Interestingly, during early differentiation, PTH transiently stimulated the expression of BMP-2 and BMP-3. The expression of genes associated with osteoblast differentiation, osteopontin, collagen type I, alkaline phosphatase and osteocalcin were markedly stimulated by PTH, as well as bone nodule formation at day 21.
These results suggest that BMP signal is necessary for initiation of the osteoblastic differentiation, and that the effect of parathyroid hormone on this process can be in part achieved via regulation of BMP gene expression.
ECTOPIC AND OTHOTOPIC OSTEOGENESIS INDUCED BY BMP-7 IN RAT
S. Zoricic*, O. Cvijanovic, I. Kristofic, I. Maric, D. Bobinac
Department of Anatomy, University of Rijeka, Rijeka, Croatia
Members of transforming growth factor-beta/bone morphogenetic protein (BMP) superfamily induce endochondral and/or intramembranous bone formation in vivo. Present study describes the efficiency BMP-7 when implanted with demineralized bone matrix carrier (IRBR) at ectopic sites in rat. Osteogenesis induced by IRBR with and without BMP-7 implanted subcutaneously in thoracic region, onto calvatial defect and intact calvariae has been studied from days 7 to 21 after implantation. Implants were separated from surrounding tissue, fixed in 4% paraformaldehyde, embedded undecalcified in MMA, cut serially at 500 microm intervals on 3-4 microm slices, and were analyzed at days 7, 14, and 21 by histologic, histochemic, histomorphometric, and immunohistochemic methods. Results showed that only IRBR implants with BMP-7 were osteoinductive. Histologic and hystomorphometric studies demonstrated that BMP-7 induced chondrogenesis at subcutaneous site and osteogenesis at calvarial site on day 7. On day 21 all impalnts consisted mainly of bone with more pronaunced osteogenesis observed in both calvarial sites. Immunohistochemic analysis revealed BMP-7 positive staining at subcutaneous sites in hypertrophic chondrocytes, infiltrate cells and fibrous membrane. BMP-3 positive staining was observed as less intense in infiltrate cells and fibrous membrane. These results suggest that BMPs induced endochondral and intramembranous osteogenesis when implanted on subcutaneous and calvarial sites, respectively.
