Biomaterials 25 (2004) 5375–5385 Stimulation of porcine bone marrow stromal cells by hyaluronan, dexamethasone and rhBMP-2 Xuenong Zoua,b,*, Haisheng Lia, Li Chenc, Anette Baatrupa, Cody B .ungera, Martin Linda a Orthopaedic Research Laboratory, Spine Section/Department of Orthopaedics, Center of Nanoscience and Biocompitability, University of Aarhus, Nørrebrogade 44, Building 1A, 8000 Aarhus C, Denmark b Department of Orthopaedics, The 5th Affiliated Hospital of Zhongshan (Sun Yat-sen) University, MeiHuaDongLu 52, 519000 Zhuhai, Guangdong, PR China c Laboratory For Stem Cell Research, University of Aalborg, Gustav Wieds Vej 10B, 8000 Aarhus C, Denmark Received10 April 2003; accepted8 December 2003 In the interest of optimizing osteogenesis in in vitro, the present study sought to determine how porcine bone marrow stromal cell (BMSc) would respond to different concentrations of hyaluronan (HY) and its different combinations with dexamethasone (Dex)andrecombinant human bone morphogenic protein-2 (rhBMP-2).
Cellular proliferation was determined by 3H-thymidine incorporation into DNA at both Days 2 and 7 when BMSc was cultivated with HY at concentrations of 0, 0.5, 1.0, 2.0 and4.0 mg/ml. HY acceleratedcellular proliferation when comparedwith cultures inthe absence of HY at both Days 2 and7. BMSc proliferation under the high HY concentration of 4 mg/ml was significantly higherthan under the other, lower HY concentrations of 0.5, 1.0 and 2.0 mg/ml.
When BMSc were cultivatedunder HY at concentrations of 0, 1.0 and4.0 mg/ml andits 12 combinations with rhBMP-2 at concentrations of 0 and10 ng/ml andDex (+, ) at both Days 2 and7, cellular responses were examinedby 3H-thymidineincorporation into DNA, cellular alkaline phosphatase (ALP) activity, and pro-collagen type I C-terminal propeptide production.
HY acceleratedcellular proliferation irrespective of the presence of Dex andrhBMP-2. HY increasedexpression of ALP activity atDay 7, whereas hadinhibitory effect at Day 2. HY andDex showedan interaction on expression of ALP acitivity irrespective of theHY dose by Day 7. Collagen synthesis was inhibited by HY irrespective of the presence of other factors at both Days 2 and 7.
When BMSc were cultivatedwith HY of 4.0 mg/ml alone, its combinations with Dex (+) and10 ng/ml rhBMP-2, andwith DMEM/FBS alone, expression of bone-relatedmarker genes was evaluatedby real-time reverse transcription-polymerase chainreaction (Real-time RT-PCR) analysis. Osteocalcin was up-regulatedunder both rhBMP-2 andHY-Dex-rhBMP-2 at Day 2, as alsounder 4 mg/ml HY, Dex, HY-Dex, Dex-rhBMP-2, and HY-Dex-rhBMP-2 by Day 7. Type 1a1 collagen was induced by rhBMP-2 onDay 2, andby Dex-rhBMP-2 on Day 7. Osteonectin andtype X collagen was only marginally inducedby HY at Day 2. Type 1a1collagen andtype X collagen were down-regulatedin the presence of 4 mg/ml HY by Day 7.
These results suggest that HY stimulates BMSc proliferation, osteocalcin gene expression, anda secretion of enzymes such as that of ALP activity in vitro. More importantly, HY can interact with Dex andrhBMP-2 to generate direct andspecific cellular effects,which couldbe of major importance in bone tissue engineering.
r 2003 Elsevier Ltd. All rights reserved.
Keywords: Cell culture; Stem cell; Hyaluronan; Dexamethasone; BMP; Bone tissue engineering nonglycan (GAG) found in all tissues and body fluids ofvertebrates as well as in some bacteria. As an almost Hyaluronic acid(HY; also calledhyaluronan) is an ubiquitous component of extracellular matrices (ECM) ancient, highly conservational, extracelluar glycosami- in long bones, HY amounts to 3% of the total GAG .
HY andproteoglycans, particularly aggrecan in theosteoidmatrix, are involvedin mineralization The *Corresponding author. Orthopaedic Research Laboratory, Aarhus distribution of hyaluronan in vitamin D-treated chick University Hospital, N rrebrogade 44, Building 1A, 8000 Aarhus C, bone andthe alterations observedin rachitic tissue Denmark. Tel.: +45-89494136; fax: +45-89494150.
E-mail address: [email protected] (X. Zou).
suggest an essential role for HY in endochondral bone 0142-9612/$ - see front matter r 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biomaterials.2003.12.041 X. Zou et al. / Biomaterials 25 (2004) 5375–5385 formation HY interacts with other macromolecules tion. Nevertheless, high molecular weight HY (900 and andplays a predominant role in tissue morphogenesis, 2300 kDa) has significantly increasedALP activity, cell migration, differentiation, and adhesion. The for- osteocalcin mRNA expression andmineralization .
Those studies did not assess, however, effects of a higher requires that mesenchymal precursors divide, differenti- concentration of HY, nor its combination with Dex ate, andmigrate to a connective tissue/HY-rich andgrowth factors on the proliferation anddifferentia- matrices. Locally appliedHY with a high molecular weight (190 kDa) has enhancednew bone formation HY (720 kDa) has excisedits multiple biologic attributes favourably in early woundhealing andtissue regenera- HY's stimulation of osteoinduction in this model is tion processes .
due, in part, to its ability to entrap and maintain The aim of the present study was to investi- endogenous BMPs and growth factors liberated from gate whether different concentrations of HY (800 kDa) bone margins of the wound. rhBMP-2 in vitro has can stimulate the proliferation of porcine BMSc ifferentiation of osteoblasts from andto evaluate the porcine BMSc response to pluripotent mesenchymal cells at only dose concentra- different combinations of HY, Dex and rhBMP-2 in tions above a specific threshold, while it has been able to an interest of optimizing osteogenesis for bone tissue induce differentiation of adipocytes at all dose concen- trations In both of animal andhuman stud rhBMP-2 has proven consistently capable of inducingnew bone formation Dexamethasone (Dex) may stimulate both uncom- 2. Materials and methods mittedstem andcommittedstromal cells. Uncommittedstem cells from fetal rat calvaria have been observedto 2.1. Cell culture system of porcine BMSc differentiate toward an osteogenic lineage when re-cruitedby Dex, presumably by lead Porcine BMSc were isolatedusing the method differentiation at the expense of growth and prolifera- described by Thomson et al. Briefly, cultures tion In the porcine BMSc cultures, Dex is sufficient derived from iliac crest bone biopsies of 3-month-old to induce the deposition of mineralized bone matrix and female Landrace pig and thus contained elements of to upregulate bone-relatedmarker genes such as bone marrow stromal cells (progenitor osteoblast osteocalcin type Ia1 collagen andosteo- cells) andtrabecular bone cells (mature osteoblast nectin in long-term cultures. The osteogenic cells). Iliac crest bone chips were harvestedaseptically potential has also been shown with neonatal porcine under general anesthesia. Bone chips with marrow BMSc. Upon incubation of media containing Dex, cells were placedinto Dulbecco's mod porcine BMSc has formedmineralizednodules, which medium with Glutamix-1, sodium pyruvate, 4500 mg/l demonstrated ALP-positive cells and a calcified type I oxine (DMEM, Gibco, BRL) con- collagen-rich matrix taining 0.1% heparin, washed, and centrifuged at HY binds to cells by direct interaction with cell 1200 rpm for 10 min at room temperature. The cells surface receptors andto extracellular matrix were collectedin 75-cm2 flasks containing 15 ml of components or to proteins such as hyaladherins DMEM supplementedwith penicillin (50 IU/ml; Sigma), , each of which produce different biologic functions streptomycin (50 mg/ml; Sigma), and10% fetal bovine in specific local environments with varying molecular serum (FBS Australian Origin; Bio Whittaker Europe, weights of HY. The cell-signaling function of HY is Belgium; Lot 8SB0001) andthereby constitutedthe mediated through CD44, which is abundantly expressed primary culture. After 24 h, the media were changed in on osteoblasts . Studies using different molecular order to remove any non-adherent cells; thereafter, the weights of HY in 1.0 and2.0 mg/ml dosages to stimulate media were changed 2 times a week. The cultures were proliferation anddifferentiation of mesenchymal stem maintainedin a humidifiedatmosphere of 95% air and cell have only recently begun to appear in the literature.
5% CO2 at 37C.
Mouse mesenchymal stem cell has showedbone colony The cells of the primary cultures reachedconfluence formation in vitro with low-molecular weights of HY after 10–14 days. The cells were then washed twice with (30 and40 kDa) in 1.0 and2.0 mg/ml concentrations, PBS buffer, after which 4 ml 0.125% trypsin/5 mm but they have not shown significant bone colony EDTA was added for 5 min at 37C in order to effect formation at a high-molecular weight of HY . Low a release. The reaction was stoppedby the addition of molecular weight HY (60 kDa) has significantly stimu- 6.0 ml of DMEM/10% FBS. After centrifugation at latedrat cell growth andosteocalcin mRNA expression 1200 rpm for 10 min at room temperature, the cells were in a dose-dependent manner, but it has shown no re-suspended in different media for the development of apparent effects on ALP activity andbone mineraliza- X. Zou et al. / Biomaterials 25 (2004) 5375–5385 2.2. Analysis effects of hyaluronan on BMSc was assayedin terms of 3H-thymidine incorporation into DNA; cellular differentiation was assayed byexpression of ALP activity in the cells andPICP BMSc's (passage1) were cultivatedin 24-well plates at a production in the media.
cell density of 13,157 cells/cm2 in 1 ml of DMEM/FBS, in DNA synthesis assay was accomplishedby a determi- both the absence of andsupplementedwith sod nation of cellular proliferation from 3H-thymidine hyaluronate (Mw=800 kDa; Lifecore Biomedical, Inc., incorporation into DNA. In all, 25 mCi/ml 3H-thymidine Chaska, MN). HY with increasing concentrations of 0.5, (0.625 mCi/well) (Lifescience, Amersham) was added to 1.0, 2.0, and4.0 mg/ml were usedin this study. A triplicate each cell layer for the final 20 h of incubation. The was obtainedper cultural med ium. Experimental cells incorporation of 3H-thymidine into trichloroacetic acid- were taken from seven individual pigs. The culture media perceptible DNA was measuredby liquidscintillogra- were changedon the 2ndand5th d phy (Beta-counter, Wallac, Finland). Intra-assay CV: proliferation was determined by 3H-thymidine incorpora- 9.4%. The result was expressedby cpm value and tion into DNA at time intervals of the 2ndand7th days.
normalizedto cultures incubatedin DMEM/FBS alone.
ALP activity was measuredin the cell layer after 2.3. BMSc response to hyaluronan, dexamethasone, and 30 min of incubation with p-nitro phenyl phosphate (Sigma) as a substrate at 37C Absorbance of p-nitro phenol was determined by micro spectrophot- BMSc response to HY, Dex, andrhBMP-2 (Genetics ometer at 405 nm. Intra-assay CV: 4.6%. Phenotype Institute, Inc., Cambridge, MA) was studied by measur- expression was estimatedby the ratio of ALP in wells ing cell proliferation andosteoblastic d supplementedwith 10% FBS. The ALP value in this BMSc's from five different pigs were cultivated in 96- study was adjusted with cell number that cells were well plates at a cell density of 6000 cells/cm2 for 2 days countedafter methylene blue staining andnormal- and3000 cells/cm2 for 7 days. They were then incubated izedto cultures incubatedin DMEM/FBS alone.
in DMEM/FBS (200 ml) with 12 different combinations In addition, histochemical staining for ALP was of HY (0, 1.0, and4.0 mg/ml), Dex/Asc/b-GP performedrandomly in supplementary cultures in order (+, ), andrhBMP-2 (0, 10 ng/ml). Dex/Asc/b-GP to test the phenotypic stability of the culture system.
examethasone (1  108 mol/l, Sigma), Pro-collagen type I C-terminal propeptide (PICP) was ascorbate (82 mg/ml, Merck), andsodium b-glyceropho- measured in the conditioned media from 96-well plates sphate (10 mmol/l, Sigma). A triplicate was obtainedper of cell culture after incubation for 2 and7 days. PICP culture medium. The culture media were changed on was quantitatedin cond Day 2 and5. After 2 and7 days, cellular proliferation commercial radio-immuno assay, using an antibody thatrecognizes procollagen C-terminal pro-peptide (PICP[125I], Orion Diagnostica, Finland). 125I radioactivitywas countedby using a gamma counter (Wallac, Turku, Table 1Twelve different kinds of culture medium (000, 100 y 211) consisting Finland). Intra-assay CV 9%. The result in this study of DMEM/FBS, supplementedwith sod ium hyaluronate (1.0 and was adjusted with cell number that cells were counted 4.0 mg/ml), dexamethasone (1  108 mol/l), ascorbate (82 mg/ml), b- after methylene blue staining andnormalizedto glycerophosphate (10 mmol/l), andrecombinant human bone morpho- cultures incubatedin DMEM/FBS alone.
genic protein-2 (10 ng/ml) 2.4. Expression of bone-related marker genes in response to hyaluronan, dexamethasone, and rhBMP-2 Bone-relatedmarker gene expression of porcine BMSc in response to hyaluronan, dexamethasone and rhBMP-2 was investigatedby real-time RT-PCR analy- sis. BMSc from 3 different pigs were cultivated in 75 cm2 flask at cell densities of 6000 cells/cm2 for 2 days and 3000 cells/cm2 for 7 days in DMEM/FBS alone and secondly in the presence of HY (4.0 mg/ml), Dex/Asc/b- GP (+), rhBMP-2 (10 ng/ml) andtheir combinations.
Real-time PCR assay was usedto quantify osteocal- cin, type 1a1 collagen, osteonetin andtype X collagen as DMEM=Dulbecco's modified Eagle's medium; FBS=fetal bovine well as GAPDH-mRNA levels. Total cellular RNA was serum; HY=sodium hyaluronate; Dex=dexamethasone; Asc=ascor-bate; b-GP=b-glycerophosphate; rhBMP-2=recombinant human extractedusing Trizol Reagent (Invitrogen, Tastrup, bone morphogenic protein-2.
Denmark). RNA samples were DNase I treatedand X. Zou et al. / Biomaterials 25 (2004) 5375–5385 Table 2Bone-relatedmarker genes Oligonucleotides (50-30) (up/down) GCT TTG CCC CGC GAT CTA ATG TTC GCC AAA TCC GTT CAC TCC GAC CTT TCA ACC CCG ACT GCG ACG AG TTG GAG CAG CTG GGA TGA TGG Type Ia1 collagen CCA AGA GGA GGG CCA AGA AGA AGG GGG GCA GAC GGG GCA GCA CTC TCC GGA TCT TTC CIT TGC TTT CTA CCT TCA CAT CGT GGC AAG AGT TTG GCC CTT TTG CTG CTG CTA TTG TC GTG TTG GAT GGT GGG CCT TTT ATG usedfor cDNA synthesis with M-MLV reverse tran- scriptase (Sigma-Aldrich Denmark A/S) and Random ðpo0:001Þ: Cellular proliferation in the presence of primers (Invitrogen, Tastrup, Denmark). The primers 4 mg/ml HY was greater than that of 0.5, 1.0 and for teat genes were designed using the Primer Select 2.0 mg/ml HY ðpo0:05Þ on Day 2 Cellular program of the Lasergene software package (DNA- proliferation in the presence of 4 mg/ml HY was greater STAR, Madison, WI). The respective sequences are than that of 0.5 and1.0 mg/ml HY ðpo0:05Þ on Day 7 listedin . Prior to routine use, the optimal (No difference was found among 0.5, 1.0 and annealing temperature andpred ictedsize of the PCR 2.0 mg/ml HY.
product for each gene was verified by gradient tests andelectrophoresis. AdvanTaq Plus DNA polymerase (BD 3.2. Effect of hyaluronan, dexamethasone and rhBMP-2 Biosciences Clontech) was usedto enable a hot-start on cell proliferation technique along with the reaction buffer recommendedby the manufacturer. To make possible the visualization BMSc was cultivatedin DMEM/FBS (200 ml) with of PCR products in real time, the SYBR Green I different combinations of HY, Dex, and rhBMP-2.
fluorophore (Molecular Probes) was usedin a final After 2 days of cultivation, concerned with the within- dilution of 22,000  from the stock supplied. A two- subject effects on cell proliferation, the effects of HY, temperature cycling, consisting of a denaturation step at rhBMP-2, andDex-HY, rhBMP-2-HY, Dex-rhBMP-2- 95C for 15 s andannealing/extension step at 60–68C HY interactions were significant ðpo0:05Þ; but the effect for 30 s was carriedout in an i-Cycler PCR system (Bio- of Dex, Dex-rhBMP-2 interaction did not reach Rad, Hercules, CA). All experiments were performed triplicate for each sample. The relative quantitative DMEM/FBS alone, cellular proliferation was signifi- expression of bone-relatedmarker genes in each sample cantly increasedin the presence of 4.0 mg/ml HY alone was normalizedto GAPDH-mRNA level. The fold andin its combinations with Dex, rhBMP-2, andthe change of gene expression was normalizedto cell culture latter two together; this was also the case using 1.0 mg/ in DMEM/FBS alone.
ml HY alone andits combination with Dex (po0:05; 2.5. Statistical analysis After 7 days of cultivation, concerned with the within- subject effects on cell proliferation, the effects of HY, Data were expressedas mean 7 standard error of the rhBMP-2, andDex-HY, rhBMP-2-HY interactions mean (SEM). Statistical analysis was performedusing were significantly different ðpo0:05Þ; but the effects of multiple analyses of variance (MANOVA) with repeated Dex-rhBMP-2 andDex-rhBMP-2-HY interactions did measures. When significant main effects or an interac- not reach significance. Comparedto cultures incubated tion between the main effects was found, specific in DMEM/FBS alone, cellular proliferation was sig- comparisons were made with paired t-tests. Statistical nificantly increasedin the presence of 4.0 mg/ml HY significance was representedby po0.05. Statistical alone andin its combinations with Dex or rhBMP-2; analysis was performedwith SPSS version 10.0 statis- this was also the case using 1.0 mg/ml HY alone andits tical software (SPSS, Chicago, IL, USA).
combinations with Dex, rhBMP-2, andthe latter twotogether (po0:05; 3.3. Effect of hyaluronan, dexamethasone and rhBMP-2on ALP activity 3.1. Effect of hyaluronan on BMSc proliferation Over a time span of 2 and7 days, ALP activity was HY significantly increasedthe incorporation of 3H- significantly increasedon Day 7 comparedto Day 2 thymidine into DNA observed at both 2 and 7 days (po0:001; After 2 days of cultivation, HY X. Zou et al. / Biomaterials 25 (2004) 5375–5385 A (Stimulation/Control) A (Stimulation/Control) H-thymidine incorporation into DN H-thymidine incorporation into DN Sodium Hyaluronate Sodium Hyaluronate Fig. 1. 3H-thymidine incorporation into DNA at increasing concentrations of 800 kDa sodium hyaluronate (0.5, 1.0, 2.0 and 4.0 mg/ml) on Day 2(A) andDay 7 (B), normalizedto cultures incubatedin DMEM/FBS.
A (Stimulation/Control) A (Stimulation/Control) H-thymidine incorporation into DN H-thymidine incorporation into DN Fig. 2. 3H-thymidine incorporation into DNA (cpm) at cultures incubated in DMEM/FBS with different combinations of HY (0, 1.0 and 4.0 mg/ml),Dex/Asc/b-GP (+, ), rhBMP-2 (0 and10 ng/ml) on Day 2 (A) andDay 7 (B), normalizedto cultures incubatedin DMEM/FBS. 000, 100 y 211are depicted in .
Alkaline Phosphatase Fig. 3. Alkaline phosphatase activity at cultures incubatedin DMEM/FBS with different combinations of HY (0, 1.0 and4.0 mg/ml), Dex/Asc/b-GP(+, ), rhBMP-2 (0 and10 ng/ml) on Days 2 and7. ALP activity were adjustedwith cell number andnormalizedto cultures incubatedin DMEM/FBS. 000, 100 y 211 are depicted in X. Zou et al. / Biomaterials 25 (2004) 5375–5385 significantly decreased ALP activity (p ¼ 0:005). In PICP was significantly decreased in the presence contrast, ALP activity was significantly increasedin cells of 4 mg/ml HY alone andits combinations with incubatedwith Dex andrhBMP-2 comparedto cultures Dex, rhBMP-2, andthe latter two together; this was incubatedin DMEM/FBS alone (p ¼ 0:04; also the case using 1 mg/ml HY in combinations Comparedto cultures incubatedin DMEM/FBS with Dex, rhBMP-2, andthe latter two together alone, after 7 days of cultivation, Dex-rhBMP-2 conditioned cultures showed higher ALP activity thandid HY-Dex conditioned cultures, when content was 3.5. Effect of hyaluronan, dexamethasone and rhBMP-2 increasedwith cells incubatedwith Dex alone andDex- on bone-relate marker gene expression rhBMP-2 ðp ¼ 0:001Þ: Comparedto cultures incubatedin DMEM/FBS alone, ALP activity was significantly Gene expression analyses of porcine BMSc cultures in increasedin the presence of Dex andrhBMP-2, 4 mg/ml DMEM/FBS alone andin the presence of 4 mg/ml HY, HY alone andthe latter's combinations with Dex; this Dex, rhBMP-2 andtheir combinations were performed was also the case using 1 mg/ml HY, Dex andrhBMP-2 for characteristic osteogenic marker genes (After together (po0:05; ).
2 days of cultivation, osteocalcin induction was in-creasedup to 29-foldin the presence of rhBMP-2 and 3.4. Effect of hyaluronan, dexamethasone and rhBMP-2 the combinations of HY-Dex-rhBMP-2 comparedwith DMEM/FBS alone. By day 7, osteocalcin was up-regulatedto 121, 32 and46-foldwhen BMSc was After 2 days and 7 day of cultivation, PICP did not cultivatedwith 4 mg/ml HY, Dex andHY-Dex, show a significant difference on Day 7 when compared respectively. However, induction of osteocalcin gene to Day 2. HY andinteractions with combinations of expression was only 6-foldby rhBMP-2, whereas Dex- Dex andrhBMP-2 significantly inhibitedcollagen type I rhBMP-2 andHY-Dex-rhBMP-2 increase 22- and13- synthesis ðpo0:05Þ: Specifically, comparedto cultures foldexpression comparedwith DMEM/FBS alone.
incubatedin DMEM/FBS alone, PICP were significant Type 1a1 collagen was raisedto an 11-foldexpression lower on Day 2 in the presence of 4 mg/ml HY alone and by rhBMP-2 on Day 2, while type 1a1 collagen was in its combinations with Dex, rhBMP-2, andthe latter causedup to 27-foldinduction with combination of Dex two together; this was also the case using 1 mg/ml HY andrhBMP-2 on Day 7, when comparedwith DMEM/ alone andits combinations with Dex andrhBMP-2 FBS alone. Osteonectin andtype X collagen was only marginally induced by HY at Day 2. Type 1a1 collagen Comparedto cultures incubatedin DMEM/FBS andtype X collagen were d own-regulatedin the alone, after time intervals of cultivation, on Day 7, presence of 4 mg/ml HY by Day 7.
ype I C-terminal Propeptide (Stimulation Control) Fig. 4. Pro-collagen type I C-terminal propeptide (PICP) at cultures incubated in DMEM/FBS with different combinations of HY (0, 1.0 and4.0 mg/ml), Dex/Asc/b-GP (, +), rhBMP-2 (0 and10 ng/ml) on Days 2 and7. PICP was adjustedwith cell number andnormalizedto culturesincubatedin DMEM/FBS. 000, 100 y 211 are depicted in X. Zou et al. / Biomaterials 25 (2004) 5375–5385 Osteocalcin Gene Expression ype I Collagen Gene ExpressionT Osteonectin Gene Expression ype X Collagen Gene ExpressionT Fig. 5. Relative fold induction of bone-related marker genes in porcine BMSc cultures undergoing osteogenic differentiation. In porcine BMSccultures with DMEM/FBS alone and the addition of 4.0 mg/ml HY, Dex/Asc/b-GP (+), 10 ng/ml rhBMP-2 or their combinations on Days 2 and7,the relative quantitative expression of osteocalcin (top left), type Ia1 collagen (top right), osteonectin (bottom left) andtype X collagen (bottom right)in each sample was normalizedto GAPDH-mRAN level. The relative foldchange was normalizedto cultures incubatedin DMEM/FBS. 000, 100 y211 are depicted in In the present study, we first investigated cellular proliferation of porcine BMSc, a putative source of Bone has a high potential for self-regenerative repair, mesenchymal progenitor cells, in the presence of HY at with progenitor cells residing in both the periosteum and a low molecule weight of 800 kDa andat the different bone marrow In bone formation, BMSc in adult concentrations of 0.5, 1.0, 2.0 and4.0 mg/ml. As bone marrow stroma are believedto play an essential reportedabove, the cellular proliferation ind role because they are a major source of osteoprogenitor HY (0.5, 1 and2 mg/ml) was consistent with the study cells. In vitro, BMSc isolatedfrom bone marrow that usedrat calvaria for cell cultures, in which HY at aspirates or biopsies of rats mice , rabbits three levels of molecular weight (60, 900 and2300 kDa) pigs , andhumans have been all significantly increasedthe thymidine uptakes of cells but with dose variations It is interesting that the mal stem cells from various species have also been done distinguishing effect of HY on cellular proliferation in In those in vitro studies, dexamethasone, the present study was observed at a high concentration (4 mg/ml), but no clear dose-response difference could brought out a demonstration of osteogenic differentia- be observedat lower d oses. This effect of HY was tion in long-term cultures with an increase of ALP irrespective of the presence of other factors (rhBMP-2 or activity, a deposition of type I collagen, bone nodule Dex). The mechanisms of cell proliferation activatedby formation, andbone-relatedmarker gene expression.
HY tendto increase the volume andsurface areas for Within this complex multilineage cell system of BMSc, cell migration and cellular activities and in addition studies of environmental determinants of cell prolifera- stimulate receptor-mediated events. HY can form a tion and differentiation may be important for develop- pericellular coat aroundcells, settle into a cell-poor ing an understanding of the full differentiation potential space in a culture well, andfacilitate both cell of these cells.
detachment from its matrix and mitosis in response to X. Zou et al. / Biomaterials 25 (2004) 5375–5385 mitogenic factors such as pre-inflammatory mediators short-term cultures. Combinedwith rhBMP-2 andDex, andgrowth factors . In the context of an in vitro a high concentration of HY (4 mg/ml) induced early culture, they can fully express their replicating and gene expression of osteocalcin on Day 2 anda low dividing potential. A high concentration of HY strands concentration of HY (1 mg/ml) increasedALP activity couldprovide a much larger active surface for surround- on Day 7. Studies have reported that the extracellular ing cells, which couldbindto the cell surface CD44 matrix components andcell shape couldmod receptor, to promote cell migration to a cell-poor space.
cellular differentiation and responsiveness to growth The high concentration of HY wouldbe offering a factors HY andbasic fibroblast growth factor hyaluronic acid-rich area that could induce the migra- can act synergistically to accelerate new bone formation tory cells to release more endogenous growth factors andstimulate cell–cell interaction, resulting in faster cell It has been extensively demonstrated by the present proliferation during early stages.
study, that when porcine BMSc are grown in monolayer Extracelluar matrix molecules are involvedin both cultures with HY, the BMSc proliferate andinitially modifying cell responses to growth factors and cytokines induce gene expression of osteocalcin and osteonectin.
andin regulating cell motility, growth, andad However, a de-differentiation process took place in interactions. Porcine BMSc responded to HY and their which HY reduced pro-collagen type I synthesis and interactions with dexamethasone and rhBMP-2 resulted down-regulated the expression of type 1a1 collagen and in a change in their phenotype expression. In the type X collagen in the early phase of a cell culture over 7 absence of HY, BMSc, once plated, adhere to the days. This suggests that HY may reduce collagen bottom of the culture well andinitiate the process of deposition in the matrix in the early stage, which is condensation, which is a critical step in cellular similar to the effects of exogenous HY in decreasing differentiation. In the presence of HY in either lower woundhealing collagen andinhibiting woundscar or high concentrations, HY settles into cell-poor space formation . This may occur because BMSc is and surrounds cells from all sides, which prevents the most likely fibroblastic in appearance in the early cells from aggregating in the early stage. HY thus proliferation phase andexpression of type 1a1 collagen inhibited ALP activity on Day 2 with dose-specific mode was up-regulated in the differentiation phase after de- irrespective of other factors. As previously mentioned, proliferation, which detected by Day 24 . It is of cells in the process were replicatedthemselves.
interest that the initiation of type X collagen gene HY binds to cells by direct interaction with adhesion expression on Day 2 was reversedby Day 7 in the molecule receptors on cell membrane surfaces: such as presence of HY alone andits combination of Dex, in cluster-determinant CD44 receptor, and receptor for which type X collagen has an important role in the hyaluronate-mediated motility (RHAMM), and ICAM- mineralization process during endochondral ossification 1 (intercellular adhesion molecule-1) They Because a temporal sequence of events during thereby take part in the enhancement of cell growth, the process of cellular proliferation has observedin the differentiation, and functions related to cell adhesion, present study, in which an enhanced expression of migration, division as well as enzyme secretion, for alkaline phosphatase occurs immediately after the proliferative period, and later an increased expression HY in the present study allowed to continue its of osteocalcin, those genes then wouldactivate the processing over a periodof time, andby Day 7, a high subsequent induction of genes associated with intracel- concentration of HY (4 mg/ml) was usedto make more lular matrix maturation andmineralization when cells to condense. It produced an upregulation of collagen deposition is promoted. Investigation of endogenous cellular ALP activity and osteocalcin collagen deposition and mineralization associated with mRNA expression, which are the mature bone-related a prolongedpresence of HY is warrantedandrelevant markers for osteoblast differentiation. When combined bone tissue engineering.
with HY andDex, an interaction between HY andDex A limitation of the present study was the use of an made the expression on ALP activity and osteocalcin in vitro model that contains a heterogeneous population gene earlier than that in another study . HY thus of cells. However, this well-documented system has been showeda time-specific andconcentration-specific mode previously usedfor confirmation of the presence of of action similar to other studies Proteoglycans, osteoblast-like cells in cultures through the identification such as HY, serve their possible role of being store- of mineralisedbone nodule formation andALP staining houses for growth factors, but they may also interact cells The present experimental design did not with such factors to generate direct and specific cellular permit the assessment of mineralisedbone nod effects There may be other important interactions formation, because the process was the object of only between HY andgrowth factor in bone repair. As short-term observation (7 days).
described here, porcine BMSc treated with HY and One important property of HY couldbe that the rhBMP-2 clearly demonstrated cellular proliferation in molecule offers a three-dimensional environment for the X. Zou et al. / Biomaterials 25 (2004) 5375–5385 cultivatedcells, which wouldprovide the stimulation of differentiation into distinct mesenchymal cell lineages. DNA Cell ifferentiation through cytoskeletal interactions. Because of its unique physicochemical [6] Asahina I, Sampath TK, Hauschka PV. Human osteogenic protein-1 induces chondroblastic, osteoblastic, and/or adipocytic properties—nonimmunogenicity of the highly purified differentiation of clonal murine target cells. Exp Cell Res 1996; form—HY has already seen biomedical applications for many years. More recently, the reportedbenefits of [7] Boden SD Overview of the biology of lumbar spine fusion and exogenously basedbiomaterials have been shown for principles for selecting a bone graft substitute. Spine 2002;27(16 tissue repair purposes. Already in this field, products Suppl 1):S26–31.
employ either pure HY or derivatives of HY, which [8] Boden SD, Martin Jr GJ, Horton WC, Truss TL, Sandhu HS.
Laparoscopic anterior spinal arthrodesis with rhBMP-2 in a use cross-linking , esterification or other titanium interbody threaded cage. J Spinal Disord 1998;11: chemical modification techniques to improve their physical handling and stability characteristics. HY's [9] Burkus JK, Dorchak JD, Sanders DL. Radiographic assessment biological function on BMSc couldbe more important of interbody fusion using recombinant human bone morphoge- in the interest of bone tissue engineering.
netic protein type 2. Spine 2003;28:372–7.
[10] Burkus JK, Gornet MF, Dickman CA, Zdeblick TA. Anterior To summarize in conclusion, this study demonstrates lumbar interbody fusion using rhBMP-2 with tapered interbody that HY accelerates cellular proliferation, increases ALP cages. J Spinal DisordTechnol 2002;15:337–49.
activity andosteocalcin gene expression, andinhibits [11] Burkus JK, Heim SE, Gornet MF, Zdeblick TA. Is INFUSE pro-collagen type I synthesis andthe expression of type bone graft superior to autograft bone? An integratedanalysis of 1a1 collagen when porcine BMSc is cultivatedwith HY clinical trials using the LT-CAGE lumbar taperedfusion device.
alone or combinedwith Dex or rhBMP-2 or the later 2 J Spinal DisordTechnol 2003;16:113–22.
[12] Sandhu HS, Toth JM, Diwan AD, Seim HB 3rd, Kanim LE, together. ALP activity andthe expression of type 1a1 Kabo JM, Turner AS. Histologic evaluation of the efficacy of collagen was also increasedin the presence of Dex and rhBMP-2 comparedwith autograft bone in sheep spinal anterior rhBMP-2. More importantly, HY interactedwith Dex interbody fusion. Spine 2002;27:567–75.
andrhBMP-2 to generate d irect andspecific cellular [13] Schimandle JH, Boden SD, Hutton WC. Experimental spinal effect, which wouldbe of major importance in bone fusion with recombinant human bone morphogenetic protein-2.
Spine 1995;20:1326–37.
tissue engineering.
[14] Bellows CG, Heersche JN, Aubin JE. Determination of the capacity for proliferation anddifferentiation of osteoprogenitorcells in the presence andabsence of d examethasone. Dev Biol [15] Carlson CS, Tulli HM, Jayo MJ, Loeser RF, Tracy RP, Mann KG, Adams MR. Immunolocalization of noncollagenous bone This study was financially supported by Implexs matrix proteins in lumbar vertebrae from intact andsurgically in the United State, The menopausal cynomolgus monkeys. J Bone Miner Res 1993;8: ( 233-425-17.04.00 MP), Helga og Peter Kornings [16] Ringe J, Kaps C, Schmitt B, Buscher K, Bartel J, Smolian H, Schultz O, Burmester GR, Haupl T, Sittinger M. Porcine mesenchymal stem cells. Induction of distinct mesenchymal cell Clinical Research in University of Aarhus in Denmark.
lineages. Cell Tissue Res 2002;307:321–7.
Genetics Institute Inc., Cambridge, MA kindly provided [17] Fisher LW, Robey PG, Tuross N, Otsuka AS, Tepen DA, Esch FS, Shimasaki S, Termine JD. The Mr 24,000 phospho-protein from developing bone is the NH2-terminal propeptide ofthe alpha-1 chain of type I collagen. J Biol Chem 1987;262:13457–63.
[18] Termine JD, Kleinman HK, Whitson SW, Conn KM, McGarvey ML, Martin GR. Osteonectin, a bone-specific protein linking [1] Hjertquist SO, Vejlens L. The glycosaminoglycans of dog mineral to collagen. Cell 1981;26:99–105.
compact bone andepiphyseal cartilage in the normal state and [19] Thomson BM, Bennett J, Dean V, Triffitt J, Meikle MC, in experimental hyperparathyroidism. Calcif Tissue Res 1968; Loveridge N. Preliminary characterization of porcine bone marrow stromal cells: skeletogenic potential, colony-forming [2] Robey PG, Fedarko NS, Hefferan TE, Bianco P, Vetter UK, activity, andresponse to d examethasone, transforming growth Grzesik W, Friedenstein A, Van der Genever PG, Dickson IR.
factor beta, andbasic fibroblast growth factor. J Bone Miner Res Influence of vitamin D status on hyaluronan localization in bone.
[20] Entwistle J, Hall CL, Turley EA. HA receptors: regulators of [3] Genever PG, Dickson IR. Influence of vitamin D status on signalling to the cytoskeleton. J Cell Biochem 1996;61:569–77.
hyaluronan localization in bone. Bone 1996;18:429–35.
[21] Oksala O, Salo T, Tammi R, Hakkinen L, Jalkanen M, Inki P, [4] Sasaki, T, Watanabe C. Stimulation of osteoinduction in bone Larjava H. Expression of proteoglycans andhyaluronan during woundhealing by high-molecular hyaluronic acid woundhealing. J Histochem Cytochem 1995;43:125–35.
[22] Toole BP. Hyaluronan in morphogenesis. J Intern Med [5] Ahrens M, Ankenbauer T, Schroder D, Hollnagel A, Mayer H, Gross G. Expression of human bone morphogenetic proteins-2 or [23] Nakamura H, Kenmotsu S, Sakai H, Ozawa H. Localization of -4 in murine mesenchymal progenitor C3H10T1/2 cells induces CD44, the hyaluronate receptor, on the plasma membrane of X. Zou et al. / Biomaterials 25 (2004) 5375–5385 osteocytes andosteoclasts in rat tibiae. Cell Tissue Res 1995; [42] Abatangelo G, Cortivo R, Martelli M, Vecchia P. Cell detachment mediated by hyaluronic acid. Exp Cell Res 1982; [24] Pilloni A, BernardGW. The effect of hyaluronan on mouse intramembraneous osteogenesis in vitro. Cell Tissue Res 1998; [43] Barnhart BJ, Cox SH, Kraemer PM. Detachment variants of Chinese hamster cells. Hyaluronic acidas a mod [25] Huang L, Cheng YY, Koo PL, Lee KM, Qin L, Cheng JC, detachment. Exp Cell Res 1979;119:327–32.
Kumta SM. The effect of hyaluronan on osteoblast proliferation [44] Bourguignon LY, Lokeshwar VB, Chen X, Kerrick WG.
and differentiation in rat calvarial-derived cell cultures. J Biomed Hyaluronic acid-induced lymphocyte signal transduction and Mater Res 2003;66A:880–4.
HA receptor (GP85/CD44)-cytoskeleton interaction. J Immunol [26] Orlidge A, D'Amore PA. Cell specific effects of glycosaminogly- cans on the attachment andproliferation of vascular wall [45] Lokeshwar VB, Iida N, Bourguignon LY. The cell adhesion components. Microvasc Res 1986;31:41–53.
molecule, GP116, is a new CD44 variant (ex14/v10) involvedin [27] Majeska RJ, Rodan GA. The effect of 1,25(OH)2D3 on alkaline hyaluronic acid binding and endothelial cell proliferation. J Biol phosphatase in osteoblastic osteosarcoma cells. J Biol Chem [46] McCourt PA, Ek B, Forsberg N, Gustafson S. Intercellular [28] Oliver MH, Harrison NK, Bishop JE, Cole PJ, Laurent GJ. A adhesion molecule-1 is a cell surface receptor for hyaluronan.
rapidandconvenient assay for counting cells culturedin J Biol Chem 1994;269:30081–4.
microwell plates: application for assessment of growth factors.
[47] Bertolami CN, Messadi DV.
J Cell Sci 1989;92:513–8.
in hardandsoft tissue repair. Crit Rev Oral Biol Med1994;5: [29] Solchaga LA, Cassiede P, Caplan AI. Different response to osteo-inductive agents in bone marrow- and periosteum- [48] Ben-Ze'ev A. Animal cell shape changes andgene expression.
[49] Nathan C, Sporn M. Cytokines in context. J Cell Biol 1991; [30] Maniatopoulos C, Sodek J, Melcher AH. Bone formation in vitro by stromal cells obtainedfrom bone marrow of young adult rats.
[50] Radomsky ML, Thompson AY, Spiro RC, Poser JW. Potential Cell Tissue Res 1988;254:317–30.
role of fibroblast growth factor in enhancement of fracture [31] Phinney DG, Kopen G, Isaacson RL, Prockop DJ. Plastic healing. Clin Orthop 1998;355(Suppl):S283–93.
adherent stromal cells from the bone marrow of commonly used [51] Wang JS, Aspenberg P. Basic fibroblast growth factor promotes strains of inbred mice: variations in yield, growth, and differ- entiation. J Cell Biochem 1999;72:570–85.
[32] Howlett CR, Cave J, Williamson M, Farmer J, Ali SY, Bab I, [52] Laurent C, Hellstrom S, Stenfors LE. Hyaluronic acidreduces Owen ME. Mineralization in in vitro cultures of rabbit marrow connective tissue formation in middle ears filled with absorbable stromal cells. Clin Orthop 1986;213:251–63.
gelatin sponge: an experimental study. Am J Otolaryngol [33] Owen ME, Cave J, Joyner CJ. Clonal analysis in vitro of osteogenic differentiation of marrow CFU-F. J Cell Sci [53] Longaker MT, Chiu ES, Adzick NS, Stern M, Harrison MR, Stern R. Studies in fetal wound healing. V. A prolonged presence [34] Haynesworth SE, Goshima J, Goldberg VM, Caplan AI.
of hyaluronic acidcharacterizes fetal woundfluid Characterization of cells with osteogenic potential from human marrow. Bone 1992;13:81–8.
[54] West DC, Shaw DM, Lorenz P, Adzick NS, Longaker MT.
[35] RickardDJ, Kassem M, Hefferan TE, Sarkar G, Spelsberg TC, Fibrotic healing of adult and late gestation fetal wounds Riggs BL. Isolation andcharacterization of osteoblast precursor correlates with increasedhyaluronidase activity andremoval of cells from human bone marrow. J Bone Miner Res 1996;11: hyaluronan. Int J Biochem Cell Biol 1997;29:201–10.
[55] Estrada LE, Dodge GR, Richardson DW, Farole A, Jimenez SA.
[36] Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Characterization of a biomaterial with cartilage-like properties Mosca JD, Moorman MA, Simonetti DW, Craig S, Marshak DR.
expressing type X collagen generatedin vitro using neonatal Multilineage potential of adult human mesenchymal stem cells.
porcine articular andgrowth plate chondrocytes. Osteoarthritis [37] Vilamitjana-Amedee J, Bareille R, Rouais F, Caplan AI, [56] Thomas JT, Boot-Handford RP, Grant ME. Modulation HarmandMF. Human bone marrow stromal cells express an of type X collagen gene expression by calcium beta-glyceropho- osteoblastic phenotype in culture. In Vitro Cell Dev Biol Anim sphate andlevamisole: implications for a possible role for type X collagen in endochondral bone formation. J Cell Sci 1990;95: [38] Hicok KC, Thomas T, Gori F, RickardDJ, Spelsberg TC, Riggs BL. Development andcharacterization of conditionally immorta- [57] Chiu KM, Keller ET, Crenshaw TD, Gravenstein S. Carnitine lizedosteoblast precursor cell lines from human bone marrow and dehydroepiandrosterone sulfate induce protein synthesis in stroma. J Bone Miner Res 1998;13:205–17.
[39] Jaiswal AI, Croft M. CD40 ligandinduction on T cell subsets by peptide-presenting B cells: implications for development [58] Ghosh P, Guidolin D. Potential mechanism of action of intra- of the primary T andB cell response. J Immunol 1997;159: articular hyaluronan therapy in osteoarthritis: are the effects molecular weight dependent? Semin Arthritis Rheum 2002;32: [40] Brecht M, Mayer U, Schlosser E, Prehm P. Increasedhyaluronate synthesis is requiredfor fibroblast d [59] Hunt DR, Jovanovic SA, Wikesjo UM, Wozney JM, Bernard Biochem J 1986;239:445–50.
GW. Hyaluronan supports recombinant human bone morphoge- [41] Mian N. Analysis of cell-growth-phase-relatedvariations in netic protein-2 induced bone reconstruction of advanced alveolar hyaluronate synthase activity of isolatedplasma-membrane ridge defects in dogs. A pilot study. J Periodontol 2001;72:651–8.
fractions of culturedhuman skin fibroblasts. Biochem J [60] Girotto D, Urbani S, Brun P, Renier D, Barbucci R, Abatangelo G. Tissue-specific gene expression in chondrocytes grown on X. Zou et al. / Biomaterials 25 (2004) 5375–5385 [62] Lisignoli G, Zini N, Remiddi G, Piacentini A, Puggioli A, Trimarchi C, Fini M, Maraldi NM, Facchini A. Basic fibroblast [61] Kim HD, Valentini RF. Retention andactivity of BMP-2 in growth factor enhances in vitro mineralization of rat bone hyaluronic acid-based scaffolds in vitro. J Biomed Mater Res marrow stromal cells grown on non-woven hyaluronic acidbased polymer scaffold. Biomaterials 2001;22:2095–105.


DOSSIER DE PRENSA La madrugada del 16 de noviembre de 1989, un destacamento de soldados salvadoreños del batallón Atlacatl entró en la Universidad Centroamericana (UCA) de San Salvador con la intención de matar a los jesuitas que vivían en la residencia de la Universidad, principalmente Ignacio Ellacuría, Segundo Montes y Ignacio Martín-Baró. Las órdenes militares indicaban que no podían quedar

Alexandre Cesar Santos AVALIAÇÃO DO TESTE PARA O GENE 3 DO CÂNCER DE PRÓSTATA (PCA3) PARA MELHOR INDICAÇÃO DE REBIÓPSIAS EM PACIENTES SUSPEITOS DESTE TUMOR. Dissertação apresentada ao Programa de Pós- Graduação da Fundação Pio XII - Hospital de Câncer de Barretos para obtenção do Título de Mestre em Ciências da Saúde.