MEHMET İBRAHİM TUĞLU, Feyzan Özdal KURT, HÜSEYİN KOCA, Aydın SARAÇ, Turgay BARUT, Aycan KAZANÇ

The contribution of differentiated bone marrow stromal stem cell-loaded biomaterial to treatment in critical size defect model in rats

Mandibular kırıklar iyileşmelerindeki zorluklar ve gelişen komplikasyonlar nedeni ile çene cerrahisinde önemli sorunlardır. Son zamanlarda gelişen biyomühendislik uygulamaları aynı zamanda ilerleyen kök hücre çalışmaları bu kırıklarda biyomateryal aracılığı ile kök hücre tedavisinin mümkün olabileceğini göstermektedir. Bu çalışmada biyomateryal üzerindeki farklılaştırılmış kök hücrelerin kırık tedavisinde etkinliği ayrıca oluşan oksidatif stres ve apoptosis ile ilişkisi araştırıldı. Erişkin Wistar sıçanların mandibulalarında 4 mm çapında yuvarlak defekt oluşturuldu. Defekt alanına hidroksi apatit jel ile hidroksi apatit jel üzerinde osteoblasta farklılaştırılmış kemik iliği stromal kök hücreleri yerleştirildi. 6 haftalık iyileşme periyodunu takiben öldürülen deneklerde alınan örnekler histolojik, immunohistokimyasal, radyolojik ve morfometrik yöntemler ile körleme olarak incelendi. Her iki uygulamada da hiçbir işlem yapılmamış veya sadece hücre konmuş kontrol kaviteleri ile karşılaştırıldığında oldukça anlamlı (P

Anahtar Kelimeler:

sıçan, kemik iliği, kemik kırıkları, hayvan modelleri, programlanmış hücre ölümü, kemik iliği hücreleri, kemik iliği nakli, immünohistokimya, alt çene kemiği

Biyomateryal üzerinde farklılaşmış kemik iliği stromal kök hücrelerinin kritik hacim Defektli sıçan modelinde uygulanmasının tedavideki yeri

Mandibular fractures present a challenge in maxillo-facial surgery due to difficulties in healing and complications. In recent years, advances in bio-engineering as well as stem-cell studies suggest that it may be possible to treat these fractures by stem cell treatment with biomaterials. In the present study, we explored the efficacy of differentiated stem cells placed on biomaterials on fracture treatment and its relation with oxidative stress and apoptosis. A 4 mm circular defect was made on the mandibulae of 20 adults Wistar rats. Hydroxyapatite gel (control, n=5) and bone marrow stromal cells differentiated into osteoblast-seeded hydroxyapatite gel (n=5) were implanted within these defects. We were also used empty cavities (n=5) and cavities filled with only cells (n=5) for negative controls. Animals were sacrificed after a 6-week healing period and samples were examined blindly by histological, immunohistochemical, radiological and morphometric methods. Compared to the control cavities that underwent no procedure or filled with just cells, there were significant (P<0.001) healings in both groups. Hydroxyapatite gel with differentiated stem cells on, however, yielded significantly (P<0.05) better new bone formation and osteoid production decreased fibrous tissue and increased cellular activity. Differentiated stromal cells combined with biomaterial accelerated the treatment in defects of critical volume within a 6-week period of healing, activated and resulted in significant formation of bone of higher quality. Promotion of bone formation by the helps of bioengineering and stromal cells has gained importance in the treatment and reconstruction of fractures.

Keywords:

rats, bone marrow, bone fractures, animal models, apoptosis, bone marrow cells, bone marrow transplant, immunohistochemistry, mandible,

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1. Lombardi LJ, Cleri DJ, Zambito RF: Osteomyelitis. In, Immunology and Infectious Diseases of the Mouth, Head, and Neck. St. Louis, MO: Mosby, 114-131, 1991.
2. Kaban LB, Glowacki J: Induced osteogenesis in the repair of experimental mandibular defects in rats. J Dent Res, 60 (7): 1356-1364, 1981.
3. Sweeney TM, Opperman LA, Persing JA: Repair of critical size defects using extracellular matrix protein gels. J Neurosurg, 83, 710-715, 1995.
4. Schmitz JP, Hollinger JO: The critical size defect as an experimental model for craniomandibular nonunions. Clin Orthop, 205, 299-308, 1986.
5. Hollinger JO, Kleinschmidt JC: The critical size defect as an experimental model to test bone repair materials. J Craniofac Surg, 1 (1): 60-68, 1990.
6. Kaban LB, Glowacki J, Murray JE: Repair of experimental mandibular bony defects in rats. Surg Forum, 30, 519-521, 1979.
7. Reddi AH: Initiation of fracture repair by bone morphogenetic proteins. Clin Orthop, 355S, S66-S72, 1998.
8. Vacanti CA, Kim W, Upton J, Vacanti MP, Mooney D, Schloo B, Vacanti JP: Tissue-engineered growth of bone and cartilage. Transplant Proc, 25 (1): 1019-1021, 1993.
9. Bab I, Gazit D, Muhlrad A, Shteyer A: Regenerating bone marrow produces a potent growth-promoting activity to osteogenic cells. Endocrinology, 123, 345-352, 1988.
10. Bruder SP, Fink DJ, Caplan AI: Mesenchymal stem cells in bone development, bone repair, and skeletal regeneration therapy. J Cell Biochem, 56, 283-294, 1994.
11. Bruder SP, Horowitz MC, Mosca JD, Haynesworth SE: Monoclonal antibodies reactive with human osteogenic cell surface antigens. Bone, 21, 225-235, 1997.
12. Wlodarski KH: Properties and origin of osteoblasts. Clin Orthop Rel Res, 252, 276-293, 1990.
13. Caplan AI: Mesenchymal stem cells. J Orthop Res, 9, 641650, 1991
14. Li IWS, Cheifetz S, McCulloch CAG, Sampath KT, Sodek J: Effects of osteogenic protein-1 (OP-1, BMP-7) on bone matrix protein expression by fetal rat calvarial cells are differentiation stage specific. J Cell Physiol, 169, 115-125, 1996.
15. Lian JB, Stein GS: The developmental stages of osteoblast growth and differentiation exhibit selective responses of genes to growth factors (TGFb1) and hormones (vitamin D and glucocorticoids). J Oral Implant, 19, 95-105, 1993.
16. Shi S, Kirk M, Kahn AJ: The role of type I collagen in the regulation of the osteoblast phenotype. J Bone Min Res, 11, 1139-1145, 1996.
17. Bruder SP, Jaiswal N, Haynesworth SE: Growth kinetics, self-renewal, and the osteogenic potential of purified human mesenchymal stem cells during extensive subcultivation and following cryopreservation. J Cell Biochem, 64, 278-294, 1997.
18. Holmes RE, Bucholz RW, Mooney V: Porous hydroxyapatite as a bone graft substitute in diaphyseal defects: A histometric study. J Orthop Res, 5, 114-121, 1987.
19. Hollinger JO, Brekke J, Gruskin E, Lee D: Role of bone substitutes. Clin Orthop, 324, 55-65, 1996.
20. Johnson KD, Frierson KE, Keller TS, Cook C, Scheinberg R, Zerwekh J, Meyers L, Sciadini MF: Porous ceramics as bone graft substitutes in long bone defects: A biomechanical, histological, and radiographic analysis. J Orthop Res, 14, 351369, 1996.
21. Saito N, Okada T, Horiuchi H, Murakami N, Takahashi J, Nawata M, Ota H, Miyamoto S, Nozaki K, Takaoka K: Biodegradable poly-D,L-lactic acid-polyethylene glycol block copolymers as a BMP delivery system for inducing bone. J Bone Joint Surg Am, 83-A Suppl 1 (Pt 2): S92-S98, 2001.
22. Solchaga LA, Dennis JE, Goldberg VM, Caplan AI: Hyaluronic acid-based polymers as cell carriers for tissue- engineered repair of bone and cartilage. J Orthop Res, 17 (2): 205-213, 1999.
23. Sayer M, Stratilatov AD, Reid J, Calderin L, Stott MJ, Yin X, MacKenzie M,Smith TJ, Hendry JA, Langstaff SD: Structure and composition of silicon-stabilized tricalcium phosphate. Biomaterials, 24 (3): 369-382, 2003.
24. Bruder SP, Kurth AA, Shea M, Hayes WC, Jaiswal N, Kadiyala S: Bone regeneration by implantation of purified, culture-expanded human mesenchymal stem cells. J Orthop Res, 16, 155-162, 1998.
25. Kadiyala S, Young RG, Thiede MA, Bruder SP: Culture expanded canine mesenchymal stem cells possess osteochondrogenic potential in vivo and in vitro. Cell Transplant, 6, 125-134, 1997.
26. Maniatopoulos C, Sodek J, Melcher A: Bone formation in vitro by stromal cells obtained from bone marrow of young adult rats. Cell Tissue Res, 254, 317-330, 1988.
27. Ishaug SL, Crane GM, Miller MJ, Yasko AW, Yaszemski MJ, Mikos AG: Bone formation by three-dimensional stromal osteoblast culture in biodegradable polymer scaffolds. J Biomed Mater Res, 36, 17-28, 1997.
28. Deliloglu-Gürhan SI, Tuğlu I, Vatansever HS, Özdal-Kurt F, Ekren H, Taylan M, Sen BH: The effect of osteogenic medium on the adhesion of rat bone marrow stromal cell (BMSC) to the hydroxyapatite (HA). Saudi Med J, 27 (3): 305311, 2006.
29. Deliloglu-Gürhan SI, Vatansever HS, Özdal-Kurt F, Tuğlu I: Characterization analysis of osteoblast derived from bone marrow in modified culture system. Acta Histochem, 108, 4957, 2006.
30. Porter RM, Huckle WR, Goldstein AS: Effect of dexamethasone withdrawal on osteoblastic differentiation of bone marrow stromal cells. J Cell Biochem, 90 (1): 13-22, 2003.
31. Liu P, Oyajobi BO, Russell RG, Scutt A: Regulation of osteogenic differentiation of human bone marrow stromal cells:interaction between transforming growth factor-beta and 1,25(OH)(2) vitamin D(3) in vitro. Calcif Tissue Int, 65 (2): 173-180, 1999.
32. Ripamonti U, Ma S, Cunningham NS, Yeates L, Reddi AH: Initiation of bone regeneration in adult baboons by osteogenin, a bone morphogenetic protein. Matrix, 12 (5): 369-380, 1992.
33. Langstaff S, Sayer M, Smith TJ, Pugh SM: Resorbable bioceramics based on stabilized calcium phosphates. Part II: Evaluation of biological response. Biomaterials, 22 (2): 135150, 2001.
34. Arosarena OA, Falk A, Malmgren L, Bookman L, Allen MJ, Schoonmaker J, Tatum S, Kellman R: Defect repair in the rat mandible with bone morphogenic proteins and marrow cells. Arch Facial Plast Surg, 5 (1): 103-108, 2003.
35. Topcu I, Vatansever S, Var A, Cavus Z, Cilaker S, Sakarya M: The effect of Misoprostol, a prostaglandin E1 analog, on apoptosis in ischemia-reperfusion-induced intestinal injury. Acta Histochem, 109 (4): 322-329, 2007.
36. Ergün G, Aktaş S: ANOVA modellerinde kareler toplamı yöntemlerinin karşılaştırılması. Kafkas Univ Vet Fak Derg, 15 (3): 481-484, 2009.
37. Dupoirieux L, Pourquier D, Picot MC, Neves M: Comparative studyof three different membranes for guided bone regeneration of rat cranial defects. Int J Oral Maxillofac Surg, 30, 58-62, 2002.
38. Beirne OR: Comparison of complication after bone removal from the lateral and medial plates of the ilium for mandibular augmentation. Int J Oral Surg, 15, 267-270, 1986.
39. Cancedda R, Mastrogiacomo M, Bianchi G, Derubeis A, Muraglia A, Quarto R: Bone marrow stromal cells and their use in regenerating bone. Novartis Foundation Symposium, 249, 133-143, 2003.
40. Yamamoto M, Tabata Y, Hong L, Miyamoto S, Hashimoto N, Ikada Y: Bone regeneration bytransformin g growth factor b1 released from a biodegradable hydrogel. J Control Rel, 64, 133-142, 2000.
41. Hong L, Tabata Y, Miyamoto S, Yamamoto M, Yamada K, Hashimoto N, Ikada Y: Bone regeneration at rabbit skull defects treated with transforming growth factor-b1 incorporated into hydrogels with different levels of biodegradability. J Neurosurg, 92, 315-325, 2000.
42. Mastrogiacomo M, Corsi A, Francioso E, Di Comite M, Monetti F, Scaglione S, Favia A, Crovace A, Bianco P, Cancedda R: Reconstruction of extensive long bone defects in sheep using resorbable bioceramics based on silicon stabilized tricalcium phosphate. Tissue Eng, 12 (5): 12611273, 2006.
43. Koerten HK, van der Meulen J: Degradation of calcium phosphate ceramics. J Biomed Mater Res, 44 (1): 78-86, 1999.
44. Bell R, Beirne OR: Effect of hydroxyappatite, tricalcium phosphate, and collagen on healing of defects in the rat mandible. J Oral Maxillofac Surg, 46, 589-594, 1988.
45. Okumura M, Ohgushi H, Dohi Y, Katuda T, Tamai S, Koerten HK, Tabata S: Osteoblastic phenotype expression on the surface of hydroxyapatite ceramics. J Biomed Mater Res, 37 (1): 122-129, 1997.
46. Anada T, Kumagai T, Honda Y, Masuda T, Kamijo R, Kamakura S, Yoshihara N, Kuriyagawa T, Shimauchi H, Suzuki O: Dose-dependent osteogenic effect of octacalcium phosphate on mouse bone marrow stromal cells. Tissue Eng, Part A, 14 (6): 965-978, 2008.
47. Fialkov JA, HolyCE, Shoichet MS, Davies JE: In vivo bone engineering in rabbit femur. J Craniofac Surg, 14, 324-332, 2003.
48. Włodarski KH, Włodarski PK, Galus R: Bioactive composites for bone regeneration. Ortop Traumatol Rehabil, 10 (3): 201-210, 2008.
49. Tracy BM, Doremus RH: Direct electron microscopy study of the bone-hydroxyapatite interface. J Biomed Mater Res, 18, 719-726, 1994.
50. Arosarena OA, Collins WL: Defect repair in the rat mandible with bone morphogenetic protein 5 and prostaglandine E1. Arch Otolaryngol Head Neck Surg, 129, 1125-1130, 2003.
51. Kadiyala S, Jaiswal N, Bruder SP: Culture-ex-panded, bone marrow-derived mesenchymal stem cells can regenerate a critical-sized segmental bone defect. Tissue Eng, 3, 173185, 1997.