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ANTICANCER RESEARCH 25: 947-954 (2005)

Osteogenic Progenitor Potency After High-Dose (COSS-96)

M. JÄGER, A. SCHULTHEIS, B. WESTHOFF and R. KRAUSPE

Department of Orthopaedics, Heinrich-Heine University Hospital Duesseldorf, D-40225 Duesseldorf, Germany

Abstract. Background: Since the first trial of chemotherapy in have demonstrated that a reduction in numbers patients with the survival rate has gradually results in diminished formation, too (1). As a improved. For more than two decades, most osteosarcoma complication of chemotherapy / corticosteroids for the patients from Germany, Austria and Switzerland have been treatment of acute lymphoblastic (ALL) and other treated according to the protocols of the Cooperative malignancies like osteosarcoma during childhood, avascular Osteosarcoma Study Group (COSS). The uniform treatment osteonecrosis appears in up to 30% of the patients (2, 3). concept of a high-dose polychemotherapy pre- and Moreover, chemotherapy may lead to a reduction of postoperatively improved the survival rate of these patients osteoprogenitor cells and affect osteoblast regeneration significantly. One severe side-effect of COSS chemotherapy is potency in and bone, which was measured by multiple osteonecrosis. Patients and Methods: In this study the a reduction of colony forming units in vitro (4). osteogenic potency of three different types was For more than two decades, most osteosarcoma patients elucidated after COSS-96 chemotherapy (high-risk ). from Germany, Austria and Switzerland have been treated Mononuclear cells were obtained from the , on protocols of the Cooperative Osteosarcoma Study Group and bone marrow of a 17-year-old female with a chondroblastic (COSS). The uniform treatment concept of a high-dose pre- osteosarcoma. The cells were cultivated for 4 weeks in standard and postoperative polychemotherapy improved the survival medium and stimulated for osteogenic differentiation after the rate of these patients significantly (5, 6). The preoperative second passage with dexamethasone, glycerolphosphate and neoadjuvant chemotherapy protocol includes the application ascorbine acid. Two weeks later, the cell cultures were analysed of doxorubicin or adriamicin, methotrexate, cisplatin and with respect to cell morphology and immunochemical stainings. ifosfamid. Results: All cells cultures showed an osteoblastic regeneration Particularly the anthracycline doxorubicin is responsible potential measured by (OC), (OP) and for bone marrow depressions during and after alkaline (ALP) expression. Compared to other chemotherapy. Its toxic effect is based on an intercalation donor tissues and localizations, the periosteum showed between the nucleotides and in inhibition of topoisomerase siginificantly higher osteoblast rates in vitro, whereas II, which results in an inhibition of DNA and RNA II, CD34 and CD45 were not expressed in any culture. synthesis, especially during the S-phase of the cell cycle (7). Conclusion: The results of this study demonstrate the survival of It can be assumed that those cells which have a high mesenchymal progenitor cells in bone marrow during COSS-96 proliferation rate, such as tumor and/or stem cells, are more polychemotherapy, which allows for an osteogenic regeneration sensitive than other cells. in vitro and potentially in vivo. Besides other tissues, multipotent mesenchymal stem cells (MSC) are localized in human bone marrow, the Clinical studies suggest that combination chemotherapy cambium of periosteal tissue (8) and in small numbers, in adversely affects bone metabolism, while in vitro studies cartilage, too (9). To date it is not known to what extent a COSS-96 chemotherapy may affect these stem cell- containing tissues. In contrast to other investigators, who developed appropriate in vitro screening systems to evaluate Correspondence to: Marcus Jäger, M.D., Department of the clinical response and efficiency of chemotherapeutics by Orthopaedics, Heinrich-Heine University Duesseldorf, Moorenstr. 5, D-40225 Duesseldorf, Germany. Tel: +49 (0)211-81-7961, Fax: cell culture systems (10, 11, 12), we elucidated the in vitro +49 (0)211-81-6281, e-mail: [email protected] regeneration potential of mesenchymal stem cells after in vivo neoadjuvant chemotherapy based on the COSS-96 Key Words: Osteosarcoma, chemotherapy, stem cell, osteoblast. protocol.

0250-7005/2005 $2.00+.40 947 ANTICANCER RESEARCH 25: 947-954 (2005)

Figure 1. Preoperative X-rays in two planes (a) and MRI scans (b) of the left lower leg showing signs of a malignant solid tumor in the proximal corresponding to an osteosarcoma.

Patients and Methods dexametasone, beta-glycerolphosphate and ascorbine acid (DAG) for another 2 weeks, as described previously by Pittenger et al. (13). Mononuclear cells were obtained from a 17-year-old female As osteoblast specific markers, (ALP), volunteer donor with a chondroblastic osteosarcoma of the left osteocalcin (OC) and osteopontin (OP) were used, whereas CD105 proximal tibia 2 months after COSS-96 polychemotherapy (high-risk antigen (endoglin) served as a marker. A von arm, doxorubicin, methotrexate, cisplatin and ifosfamid) (Figures 1 Kossa showed in vitro calcification. Furthermore collagen II and 2). All experiments were carried out under written consent was measured to detect chondroblastic differentiation, while CD34 / according to the Declaration of Helsinki for good scientific practice. CD45 served to evaluate for hematopoietic cell differentiation. The No metastases were detected by bone scan or MRI scans, but specimens were fixed with 5% paraformaldehyde at 4ÆC for 30 min, there was infiltration of the neurovascular bundle. The patient rinsed in -buffered saline (PBS) and dehydrated in graded underwent knee amputation (exarticulation) with a wide tumor-free alcohols. Endogenous peroxidases of the specimens were blocked by resection line confirmed by the pathologist. Tumor-free cartilage and 3% perhydrol-isopropanol solution. After rinsing in Tris-buffer, the periosteoum strips of 0.5 x 0.5 mm in length were obtained from the cell culture dishes were incubated with primary antibodies against ankle joint (tibia, fibula and talus of the amputate). Furthermore, CD-antigens with further incubation at 4ÆC for 12 h. For optical spongious bone marrow was taken from the distal tibia, fibula and visualization, the second antibody system with avidin-biotin-complex talus. The tissue specimens were cultivated for primary explant and 3,3-diaminobenzidine was used. ALP activity was measured by cultures in DMEM (PAA, Cölbe, FRG) with 10% FCS (FCS, direct substrate incubation (SK-5.200, Vector, Burlingame CA, Biochrome, Berlin, FRG), at 37.0ÆC and 5.0% CO2 for 2 weeks. USA) for 30 min at room temperature (RT). The cell cultures were Afterwards the strips / solid tissue samples were removed and the analysed blind by an independent observer via episcopic light adherent cells were passaged (0.05% trypsin/EDTA). The cultures microscopy (Axiovert 200, Zeiss, FRG) in combination with a were expanded for two weeks until a confluent monolayer occurred. computer imaging picture analysis system (Axiovision, Zeiss), after After the second passage, osteogenic stimulation was initiated by an in vitro follow-up of 6 weeks in total.

948 Jäger et al: Osteoprogenitor Cells After Chemotherapy

Figure 2. Chemotherapy protocol of COSS-96 scheme. A: Adriamycin or Doxorubicin (90 mg / m2 over 48 h i.v.), M: Methotrexate with Folin acid- Rescue (12 g / m2 within 4 h i. v., Folin-Rescue 24 h after Methotrexate application), I: Ifosfamid (3 g / m2 over 1 h i. v.), P: Cisplatin (120 mg / m2 over 72 h i. v.), C: Carboplatin (150 mg / mÇ over 1 h i. v.), E: Etoposid (150 mg / m2 over 1 h i. v.). Low risk: volume of tumor ≤ 150 ml in regression IÆ or IIÆ. High risk: volume of tumor > 150 ml in regression VÆ or VIÆ. Standard risk: patients with a volume of tumor ≤ 150 ml in regression IIIÆ-VIÆ or > 150 ml in regression IÆ-IVÆ.

For statistical analysis the Student’s t-test for independent Compared to other cell cultures, the cells isolated from statistical groups was used. P<0.01 was rated highly statistical the periosteum of the fibula showed the highest number of significant and p<0.05 statistically significant, whereas p>0.05 OC- and OP-positive cells, which characterize the mature showed no significance. The average values (X) and standard osteoblast. Corresponding to the potential of osteoblast deviations (SD) served as descriptive parameters. differentiation, CD105+ cells were present in all cell Results cultures. Calcification was found in all OP- and OC-positive cultures, demonstrated by von Kossa staining. Furthermore, all cultures showed no collagen II expression in The results of this study show clearly that, after COSS-96 immunocytochemistry after osteoblastic stimulation with chemotherapy, an osteogenic stem cell differentiation is DAG. Figure 3 illustrates the immunocytochemical staining maintained. By primary cell cultures derived from cartilage, behaviour for , , hematopoietic and periosteum and bone marrow in different organs, we could mesenchymal progenitor cells in vitro, whereas Figures 4 demonstrate a high osteoblast differentiation rate (Figure and 5 show the quantification of the . 3). There were significant quantitative differences in the osteogenic potential between the cell cultures of different Discussion tissues (cartilage, periosteum, bone marrow) and origins (talus, tibia, fibula) (Figure 4). After 2 weeks in vitro, all To date there has been a lack of scientific information dealing cultures showed a comparable monolayer of -like with osteoblast in vitro regeneration after COSS cells with a central nucleus. polychemotherapy in osteosarcoma patients. We observed the

949 ANTICANCER RESEARCH 25: 947-954 (2005)

Figure 3. a - g. Immuncytochemical staining against typical osteblast markers (OC, OP, v. Kossa), the marker collagen type II, the mesenchymal stem cell marker CD105 and both hematopoietic cell antigens CD34 and CD45 in a bone marrow-derived cell culture system. In contrast to a strong expression of the osteoblast markers and signs of biomineralization (a, c, e, g), neither the chondroblast collagen II (f) nor hematopoietic marker (b, d) were positive in immuncytochemical staining.

950 Jäger et al: Osteoprogenitor Cells After Chemotherapy

4A

4B

Figure 4A, B

951 ANTICANCER RESEARCH 25: 947-954 (2005)

4C

Figure 4. a - c. Quantification of antigen-positive cells by episcopic light microscopy.

highest osteoblast numbers in periosteum derived from the Furthermore, the variance of distribution in dexamethasone fibula compared to other donor sites and tissue types. Our receptors in the could be one reason for the findings correspond to those of McDuffee et al. (14), who different cellular response to an osteoblastic in vitro compared the in vitro osteogenic potential of cancellous bone stimulus within one tissue (20). of different conventional donor sites with tibial periosteum in Although we expect a lower rate of osteoblast an equine model. They found significant differences between differentiation from cultures derived from cartilage several donor regions. Tuber coxae and tibial periosteum compared to bone marrow and periosteum, we found high showed significantly greater numbers of osteoprogenitor cells numbers of osteoblasts in those cultures. One reason for than other tissues. These results were verified by Arnold et al. this phenomenon could be the absent vascularization of (15) and O’Driscoll et al. (16) who demonstrated that the cartilage, which may result in a lower local concentration of human cambium layer of the periosteum contains osteo- and cytotoxic chemotherapeutics in vivo. We found no chondroblastic progenitor cells with a donor age- and significant differences between cartilage cultures of the localisation-dependent regeneration potential. tibia, fibula and talus. Our personal clinical experiences with surgical Even in cartilage-derived cell cultures, there were no applications of periosteal flaps for autologous osteochondral collagen II-positive cells on immunocytochemistry staining regeneration in young patients with osteocartilage defect observed after 6 weeks in vitro. We hypothesize a zones after chemotherapy support these in vitro results of a dedifferentiation of chondroblasts, which may be promoted high regenerative potency in the periosteum (3). Moreover, by a lack of cartilage cellular contact, humoral stimuli and autologous fibula transplantation has been a standard cytomechanical forces. During cellular isolation from method to reconstruct bony defects in paediatric orthopaedic different tissue slides, we found a fibroblastoid cell type, patients for many years (17, 18). Based on clinical findings, a which was observed within the first 10 days. This cell type is periosteum-preserving technique for harvesting fibula typical of the morphology of mesenchymal stem cells (3). transplants in children or adolescents allows for a full Another explantion for cellular dedifferentiation could be regeneration of the fibula at the donor site (19). the phenomenon of cellular preaging in vitro. Parsch et al.

952 Jäger et al: Osteoprogenitor Cells After Chemotherapy

Figure 5. In vitro cell differentiation from joint cartilage taken from the distal tibia, trochlea of the talus and the distal fibula after osteoblastic stimulation by DAG over 21 d. Quantitative comparison between cells with OP, OC and ALP expression in dependency of their origin.

(21, 22) showed a significant loss of telomere length in References mesenchymal stem cells and chondroblasts after cultivation. 1 Davies JH, Evans BA, Jenney ME and Gregory JW: In vitro effects Kögler et al. demonstrated that a reduction rate in telomere of combination chemotherapy on osteoblasts: implication for length differs between mesenchymal progenitor cells of osteopenia in childhood malignancy. Bone 31(2): 319-326, 2002. different sources (23). 2 Raab P, Kuhl J and Krauspe R: Multifokale Osteonekrosen bei Currently there is a paucity of treatment concepts to heal Kindern und Jugendlichen nach Polychemotherapie. Z Orthop bone or cartilage and bone defects caused by osteonecrosis 135: 444-450, 1997. in patients with a low osteoblast regeneration potential 3 Werner A, Jäger M, Schmitz H and Krauspe R: Joint preserving after polychemotherapy. Corresponding to the positive surgery for osteonecrosis and osteochondral defects after chemotherapy in childhood. Klin Padiatr 215(6): 332-337, 2003. experiences in transplantation, 4 Banfi A, Podesta M, Fazzuoli L, Sertoli MR, Venturini M, Santini HLA-matched mesenchymal stem cell-based tissue G, Cancedda R and Quarto R: High-dose chemotherapy shows a engineering may be a promising alternative in the dose-dependent toxicity to bone marrow osteoprogenitors: a treatment of chemotherapy-induced mesenchymal tissue mechanism for post-bone marrow transplantation osteopenia. defects in the future. 92(9): 2419-2428, 2001.

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5 Bielack SS, Kempf-Bielack B, Delling G, Exner GU, Flege S, 15 Arnold U, Lindenhayn K and Perka C: In vitro-cultivation of Helmke K, Kotz R, Salzer-Kuntschik M, Werner M, Winkelmann human periosteum derived cells in bioresorbable polymer-TCP- W, Zoubek A, Jurgens H and Winkler K: Prognostic factors in composites. Biomaterials 23(11): 2303-2310, 2002. high-grade osteosarcoma of the extremities or trunk: an analysis 16 O'Driscoll SW and Fitzsimmons JS: The role of periosteum in of 1,702 patients treated on neoadjuvant cooperative osteosarcoma cartilage repair. Clin Orthop 391 Suppl: S190-207, 2001. study group protocols. J Clin Oncol 20(3): 776-790, 2002. 17 Immenkamp M, Ehrenbrink H and Knoche U: Subperiosteal 6 Ozaki T, Flege S, Kevric M, Lindner N, Maas R, Delling G, segmental resection and insertion of a fibula graft in the Schwarz R, von Hochstetter AR, Salzer-Kuntschik M, Berdel treatment of solitary bone cysts of the . Arch Orthop WE, Jurgens H, Exner GU, Reichardt P, Mayer-Steinacker R, Trauma Surg 100(2): 107-114, 1982. Ewerbeck V, Kotz R, Winkelmann W and Bielack SS: 18 Zaretski A, Amir A, Meller I, Leshem D, Kollender Y, Barnea Osteosarcoma of the : experience of the Cooperative Y, Bickels J, Shpitzer T, Ad-El D and Gur E: Free fibula long Osteosarcoma Study Group. J Clin Oncol 21(2): 334-341, 2003. bone reconstruction in orthopedic oncology: a surgical 7 Jarvinen TA, Holli K, Kuukasjarvi T and Isola JJ : Predictive algorithm for reconstructive options. Plast Reconstr Surg value of topoisomerase IIalpha and other prognostic factors for 113(7): 1989-2000, 2004. epirubicin chemotherapy in advanced breast cancer. Br J 19 Bettin D, Bohm H, Clatworthy M, Zurakowski D and Link TM: Cancer 77(12): 2267-2273, 1998. Regeneration of the donor side after autogenous fibula 8 Simon TM, Van Sickle DC, Kunishima DH and Jackson DW: transplantation in 53 patients: evaluation by dual x-ray Cambium cell stimulation from surgical release of the absorptiometry. Acta Orthop Scand 74(3): 332-336, 2003. periosteum. J Orthop Res 21(3): 470-480, 2003. 20 Abu EO, Horner A, Kusec V, Triffitt JT and Compston JE: 9 de la Fuente R, Abad JL, Garcia-Castro J, Fernandez-Miguel The localization of the functional alpha G, Petriz J, Rubio D, Vicario-Abejon C, Guillen P, Gonzalez in human bone. J Clin Endocrinol Metab 85(2): 883-889, 2000. MA and Bernad A: Dedifferentiated adult articular 21 Parsch D, Brummendorf TH, Richter W and Fellenberg J: : a population of human multipotent primitive Replicative aging of human articular chondrocytes during ex cells. Exp Cell Res 297(2): 313-328, 2004. vivo expansion. Rheum 46(11): 2911-2916, 2002. 10 Pinski J, Parikh A, Bova GS and Isaacs JT: Therapeutic 22 Parsch D, Fellenberg J, Brummendorf TH, Eschlbeck AM and implications of enhanced G(0)/G(1) checkpoint control induced Richter W: Telomere length and activity during by coculture of cells with osteoblasts. Cancer expansion and differentiation of human mesenchymal stem cells Res 61(17): 6372-6376, 2001. and chondrocytes. J Mol Med 82(1): 49-55, 2004. 11 Debes A, Rommel F, Breise M, Willers R, Gobel U and 23 Kögler G, Sensken S, Airey JA, Trapp T, Muschen M, Wessalowski R: In vitro test-system for chemo- and Feldhahn N, Liedtke S, Sorg RV, Fischer J, Rosenbaum C, thermosensitivity: an analysis of survival fractions and cell-cycle Greschat S, Knipper A, Bender J, Degistirici O, Gao J, Caplan distributions in human Ewing's as a model for tumors AI, Colletti EJ, Almeida-Porada G, Muller HW, Zanjani E and in pediatric oncology. Klin Padiatr 214(4): 223-229, 2002. Wernet P: A new human somatic stem cell from placental cord 12 Agiostratidou G, Demertzis N and Gonos ES: Evaluation of with intrinsic pluripotent differentiation potential. J Exp cytotoxic treatment of patients with osteosarcoma by an in vitro Med 200(2): 123-135, 2004. chemoresistance assay. Anticancer Res 20(5B): 3603-3608, 2000. 13 Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, Moorman MA, Simoneti DW, Craig S and Marshak DR: Multilineage potential of adult human mesenchymal stem cells. Science 284: 143-147, 1999. 14 McDuffee LA and Anderson GI: In vitro comparison of equine cancelleous bone graft donor sites and tibial perioseum as Received November 5, 2004 sources of viable osteoprogenitors. Vet Surg 32(5): 455-463, 2003. Accepted February 21, 2005

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