Bone keeps an important role in cranial skeleton and throughout the body, so bone lesions and defects are the most concerned. Although the autografts and allografts are often used to treat bone defects, they have limitations, such as cost, donor morbidity, limited osteoinductive potential, and disease transmission. In order to overcome limitations of the current therapies, bone tissue engineering has developed. It is combination of osteogenic cells, scaffolds and bone inductive factors.
Mesenchymal stem cells (MSCs) can differentiate into osteoblasts, so they become an attractive source in bone tissue engineering. Numerous studies have demonstrated the bone-building capacity of mesenchymal stem cells and even their usefulness and application in critical size bone defect treatment. Although MSCs derived from bone marrow were used in most of previous studies, limitations of using of bone marrow stem cells (BMSC) are the low frequency of osteoprogenitors in marrow, a very painful bone marrow aspiration, high donor site morbidity and the age-related decline in the number of these cells. The current studies have proved that adipose stem cells (ASCs) are abundant, easily accessible with minimal donor morbidity, and can migrate to the site of injury and have excellent expansion and proliferation capacities. ASCs have extensive osteogenic capacity both in vitro and in vivo in several species. Therefore, adipose stem cells are considered to be an attractive alternative to bone marrow stem cells.
Besides cell source, scaffold or membrane can support for bone tissue engineering. Gelatin/collagen I (GCI) membrane is known as a biocompatible and safe scaffold in tissue engineering. It is suitable to adherence and growth of adipose stem cells and can guide the differentiation of adipose stem cells in osteoblasts.
In addition to cell source and scaffold, resveratrol is used as an osteoinductive signal. It was found in grapes, peanuts and mulberries. It can enhance osteogenic capacity of adipose stem cells in bone regeneration therapy. It can also prevent cancers, extend life, reduce risks of heart disease, ameliorate common diabetes symptoms, and has anti-inflammatory and antiviral effects.
In our study, we combined human adipose stem cells (hASCs) with resveratrol-contained gelatin/collagen I (RSV/GCI) membrane to estimate biocompatibility of membranes through observation of hASCs adherence and proliferation, to assess in vitro osteogenic differentiation of hASCs on membranes by expression of osteocalcin marker and extracellular matrix mineralization, and to evaluate bone regeneration of hASC-contained RSV/GCI membrane in rat calvarial defect model. We hope that combination of hASCs and RSV/GCI membrane can bring an optimal therapy in bone tissue engineering.
In our results, we recognized the biocompatibility of membranes to human adipose stem cells clearly under the light microscope, fluorescent microscope with Hoechst nuclear staining and scanning electron microscope (SEM). Besides, the osteogenic differentiation of hASCs on membranes was proved by immuno-fluorescent staining and analyzed by flow cytometry. The osteogenic differentiation of hASCs also showed by extracellular calcium phosphate deposition of these cells on membranes, which was observed through Alizarin red staining and Von Kossa staining. Moreover, our in vivo results indicated that when we implanted the RSV/GCI membrane seeded osteogenically differentiated hASCs into rat calvarial defects for 8 weeks, the largest amount of new bone was formed on these defects. In addition to osteogenic differentiation, we approved that hASCs induced keratinocytes, neurocytes and chondrocytes on membranes without their specific inductive media by observation with immuno-staining.
In our conclusion, GCI membrane and RSV/GCI membrane have good biocompatibility. The combination hASCs and RSV/GCI membrane is an optimal strategy in osteogenic differentiation in vitro and bone regeneration in vivo. This combination can open new therapy for further clinical applications in bone tissue engineering. Besides that, GCI membrane can enhance differentiation potential of hASCs into other cell lineages such as, keratinocytes, neurocytes and chondroncytes although hASCs were not supported by specific inductive media.

Bone keeps an important role in cranial skeleton and throughout the body, so bone lesions and defects are the most concerned. Although the autografts and allografts are often used to treat bone defects, they have limitations, such as cost, donor morbidity, limited osteoinductive potential, and disease transmission. In order to overcome limitations of the current therapies, bone tissue engineering has developed. It is combination of osteogenic cells, scaffolds and bone inductive factors.
Mesenchymal stem cells (MSCs) can differentiate into osteoblasts, so they become an attractive source in bone tissue engineering. Numerous studies have demonstrated the bone-building capacity of mesenchymal stem cells and even their usefulness and application in critical size bone defect treatment. Although MSCs derived from bone marrow were used in most of previous studies, limitations of using of bone marrow stem cells (BMSC) are the low frequency of osteoprogenitors in marrow, a very painful bone marrow aspiration, high donor site morbidity and the age-related decline in the number of these cells. The current studies have proved that adipose stem cells (ASCs) are abundant, easily accessible with minimal donor morbidity, and can migrate to the site of injury and have excellent expansion and proliferation capacities. ASCs have extensive osteogenic capacity both in vitro and in vivo in several species. Therefore, adipose stem cells are considered to be an attractive alternative to bone marrow stem cells.
Besides cell source, scaffold or membrane can support for bone tissue engineering. Gelatin/collagen I (GCI) membrane is known as a biocompatible and safe scaffold in tissue engineering. It is suitable to adherence and growth of adipose stem cells and can guide the differentiation of adipose stem cells in osteoblasts.
In addition to cell source and scaffold, resveratrol is used as an osteoinductive signal. It was found in grapes, peanuts and mulberries. It can enhance osteogenic capacity of adipose stem cells in bone regeneration therapy. It can also prevent cancers, extend life, reduce risks of heart disease, ameliorate common diabetes symptoms, and has anti-inflammatory and antiviral effects.
In our study, we combined human adipose stem cells (hASCs) with resveratrol-contained gelatin/collagen I (RSV/GCI) membrane to estimate biocompatibility of membranes through observation of hASCs adherence and proliferation, to assess in vitro osteogenic differentiation of hASCs on membranes by expression of osteocalcin marker and extracellular matrix mineralization, and to evaluate bone regeneration of hASC-contained RSV/GCI membrane in rat calvarial defect model. We hope that combination of hASCs and RSV/GCI membrane can bring an optimal therapy in bone tissue engineering.
In our results, we recognized the biocompatibility of membranes to human adipose stem cells clearly under the light microscope, fluorescent microscope with Hoechst nuclear staining and scanning electron microscope (SEM). Besides, the osteogenic differentiation of hASCs on membranes was proved by immuno-fluorescent staining and analyzed by flow cytometry. The osteogenic differentiation of hASCs also showed by extracellular calcium phosphate deposition of these cells on membranes, which was observed through Alizarin red staining and Von Kossa staining. Moreover, our in vivo results indicated that when we implanted the RSV/GCI membrane seeded osteogenically differentiated hASCs into rat calvarial defects for 8 weeks, the largest amount of new bone was formed on these defects. In addition to osteogenic differentiation, we approved that hASCs induced keratinocytes, neurocytes and chondrocytes on membranes without their specific inductive media by observation with immuno-staining.
In our conclusion, GCI membrane and RSV/GCI membrane have good biocompatibility. The combination hASCs and RSV/GCI membrane is an optimal strategy in osteogenic differentiation in vitro and bone regeneration in vivo. This combination can open new therapy for further clinical applications in bone tissue engineering. Besides that, GCI membrane can enhance differentiation potential of hASCs into other cell lineages such as, keratinocytes, neurocytes and chondroncytes although hASCs were not supported by specific inductive media.

1. Rose FR and Oreffo RO. Bone tissue engineering: hope vs hype. Biochem Biophys Res Commun, 292 (2002), pp. 1-7.
2. Braddock M, Houston P, Campbell C and Ashcroft P. Born again bone: tissue engineering for bone repair. News Physiol Sci, 16 (2001), pp. 208-213.
3. Laurencin C, ASTM international. Bone Graft Substitutes (2003).
4. Pecina M, Giltaij LR and Vukicevic S. Orthopaedic applications of osteogenic protein-1 (BMP-7). Int Orthop, 25 (2001), pp. 203-208.
5. Korbling M, Estrov Z and Champlin R. Adult stem cells and tissue repair. Bone Marrow Transplant, 32 (2003), pp. S23-S24.
6. Lynn AK, Yannas IV and Bonfield W. Antigenicity and immunogenicity of collagen. J. Biomed. Mater. Res., B. Appl. Biomater, 71B (2004), pp. 343-354.
7. Veis A. The Macromolecular Chemistry of Gelatin. New York and London. Academic Press, 1994.
8. Lucie Fremont. Biological effects of resveratrol. Life Sciences, 8 (2000), pp. 663-673.
9. Sarah Sundelacruz and David Kaplan. Stem cell- and scaffold-based tissue engineering approaches to osteochondral regenerative medicine. Semin Cell Dev Biol, 20 (2009), pp. 646-655.
10. Tögel F and Westenfelder C. Adult bone marrow-derived stem cells for organ regeneration and repair. Developmental Dynamics, 236 (2007), pp. 3321-3331.
11. Nishida S, Endo N and Yamagiwa H. Number of osteoprogenitor cells in human bone marrow markedly decreases after skeletal maturation. J Bone Miner Metab, 17 (1999), pp. 171-177.
12. Gronthos S. Surface protein characterization of human adipose tissue-derived stromal cells. Cell. Physiol, 189 (2001), pp. 54–63.
13. Gimble JM, Katz AJ, and Bunnell BA. Adipose-derived stem cells for regenerative medicine. Circulation Research, 100 (2007), pp. 1249-1260.
14. Monaco E, Bionaz M, Hollister SJ and Wheeler MB. Strategies for regeneration of the bone using porcine adult adipose-derived mesenchymal stem cells. Theriogenology, 75 (2011), pp. 1381-1399.
15. Blanc Le, Tammik C, Rosendahl K, Zetterberg E and Ringden O. HLA expression and immunologic properties of differentiated and undifferentiated mesenchymal stem cells. Exp Hematol, 31 (2003), pp. 890-896.
16. Mesimäki K, Lindroos B and Törnwall J. Novel maxillary reconstruction with ectopic bone formation by GMP adipose stem cells. International Journal of Oral and Maxillofacial Surgery, 38 (2009), pp. 201-209.
17. Karageorgiou V and Kaplan D. Porosity of 3D biomaterial scaffolds and osteogenesis. Biomaterials, 26 (2005), pp. 5474-5491.
18. Zeltinger J, Sherwood JK, Graham DA, Mèueller R and Griffith LG. Effect of pore size and void fraction on cellular adhesion, proliferation, and matrix deposition. Tissue Eng, 7 (2001), pp. 557-572.
19. Chunlin Y, Hillas PJ, Buez JA, Nokelainen M, Balan J, Tang J, Spiro R and Polarek JW. The application of recombinant human collagen in tissue engineering. BioDrugs, 18 (2004), pp. 103-119.
20. Patrícia Malafaya, Gabriela Silva and Rui Reis. Natural–origin polymers as carriers and scaffolds for biomolecules and cell delivery in tissue engineering applications. Advanced Drug Delivery Reviews, 59 (2007), pp. 207–233.
21. Davidson PF, Levine L, Drake MP, Rubin A and Bump S. The serologic specificity of tropocollagen telopeptides. J. Exp. Med, 126 (1997), pp. 331-346.
22. Friess W. Collagen: biomaterial for drug delivery. Eur. J. Pharm. Biopharm, 45 (1998), pp. 113-136.
23. Olsen D, Yang C, Bodo M, Chang R, Leigh S, Baez J, Carmichael D, Perala M, Hamalainen E, Jarvinen M and Polarek J. Recombinant collagen and gelatin for drug delivery. Adv. Drug Deliv. Rev, 55 (2003), pp. 1547-1567.
24. Ross-Murphy SB. Structure and rheology of gelatin gels: recent progress. Polymer, 33 (1999), pp. 2622-2627.
25. Malda J, Kreijveld E, Temenoff JS, Blitterswijk C and Riesle J. Expansion of human nasal chondrocytes on macroporous microcarriers enhances redifferentiation. Biomaterials, 24 (2003), pp. 5153-5161.
26. Alarcón C, Lastra1 C and Villegas I. Resveratrol as an antioxidant and pro-oxidant agent: mechanisms and clinical implications. Biochem Soc Trans, 35 (2007), pp. 1156-1160.
27. Losa GA. Resveratrol modulates apoptosis and oxidation in human blood mononuclear cells. Eur J Clin Invest, 33 (2003), pp. 818-823.
28. Jang M, Cai L, Udeani GO, Slowing KV, Thomas CF, Beecher CW, Fong HH, Farnsworth NR, Kinghorn AD, Mehta RG and Moon RC. Cancer chemopreventive activity of resveratrol, a natural product derived from grapes. Science. 275 (1997), pp. 218-220.
29. Gentilli M, Mazoit JX, Bouaziz H, Fletcher D, Casper RF, Benhamou D and Savouret JF. Resveratrol decreases hyperalgesia induced by carrageenan in the rat hind paw. Life Sci. 68 (2001), pp. 1317-21.
30. Boissy P, Andersen TL, Abdallah BM, Kassem M, Plesner T and Delaisse JM. Resveratrol inhibits myeloma cell growth, prevents osteoclast formation, and promotes osteoblast differentiation. Cancer Res, 65 (2005), pp. 9943-9952.
31. Clerc D, Fermand JP and Mariette X. Treatment of multiple myeloma. Joint Bone Spine, 70 (2003), pp. 175-186.
32. Szpalski C, Barr J, Wetterau M, Saadeh PB and Warren SM. Cranial bone defects: current and future strategies. Neurosurg Focus, 29 (2010), E8.
33. Aalami OO, Nacamuli RP, Lenton KA, Cowan CM, Fang TD and Fong KD. Applications of a mouse model of calvarial healing: differences in regenerative abilities of juveniles and adults. Plast Reconstr Surg, 114 (2004), pp. 713-720.
34. Schmitz JP, Schwartz Z, Hollinger JO and Boyan BD. Characterization of rat calvarial nonunion defects. Acta Anat (Basel), 138 (1990), pp. 185-192.
35. Szpalski C, Barr J, Wetterau M, Saadeh PB and Warren SM. Cranial bone defects: current and future strategies. Neurosurg Focus, 29 (2010), pp. E8.
36. Monaco E, Bionaz M, Hollister SJ and Wheeler MB. Strategies for regeneration of the bone using porcine adult adipose-derived mesenchymal stem cells. Theriogenology, 75 (2011), pp. 1381-1399.
37. Robert L. Matrix biology: past, present and future. Pathol. Biol., 49 (2001), pp. 279-283.
38. Lee CH, Singla A and Lee Y. Biomedical applications of collagen. Int. J. Pharm, 221 (2001), pp. 1-22.
39. Rebelatto CK, Aguiar AM, Moretão MP, Senegaglia AC, Hansen P, Barchiki F, Oliveira J, Martins J, Kuligovski C, Mansur F and Christofis A. Dissimilar differentiation of mesenchymal stem cells from bone marrow, umbilical cord blood, and adipose tissue. Exp Biol Med (Maywood), 233 (2008), pp. 901-13.
40. Zhang YS, Gao JH and Lu F. Cellular compatibility of type collagen I scaffold and human adipose-derived stem cells. Nan Fang Yi Ke Da Xue Xue Bao, 27 (2007 Feb), pp. 223-225.
41. Yang C, Frei H, Rossi FM and Burt HM. The differential in vitro and in vivo responses of bone marrow stromal cells on novel porous gelatin-alginate scaffolds. J Tissue Eng Regen Med, 3 (2009), pp. 601-614.
42. Feng Zhao, Warren Grayson, Teng Ma, Bruce Bunnell and William Lu. Effects of hydroxyapatite in 3-D chitosan–gelatin polymer network on human mesenchymal stem cell construct development. Biomaterials, 27 (2006), pp. 1859-1867.
43. Zangrossi S, Marabese M and Broggini M. Oct-4 expression in adult human differentiated cells challenges its role as a pure stem cell marker. Stem Cells, 25 (2007), pp. 1675-80
44. Lee NK, Sowa H, Hinoi E, Ferron M, Ahn JD, Confavreux C, Dacquin R, McKee MD, Jung DY, Zhang Z, Kim JK, Mauvais-Jarvis F and Ducy P. Endocrine regulation of energy metabolism by the skeleton. Cell, 130 (2007), pp. 456-469.
45. Mirjam Fröhlich, Warren Grayson, Darja Marolt, Jeffrey Gimble and Nevenka Kregar-Velikonja. Bone Grafts Engineered from Human Adipose-Derived Stem Cells in Perfusion Bioreactor Culture. Tissue Engineering, 16 (2010), pp. 179-189.
46. Gastaldi G, Asti A, Scaffino MF, Visai L, Saino E and Cometa AM. Human adipose-derived stem cells (hASCs) proliferate and differentiate in osteoblast-like cells on trabecular titanium scaffolds. J Biomed Mater Res A, 94 (2010), pp. 790-799.
47. Christopher Paul Erdman. Enrichment of Adipose-derived Mesenchymal Stem Cells Using Resveratrol. A Thesis Presented to the Academic Faculty. Department of Biomedical Engineering, Georgia Institute of Technology, 2009.
48. Plant A and Tobias JH. Characterization of the temporal sequence of osteoblast gene expression during estrogen-induced osteogenesis in female mice. J Cell Biochem, 82 (1999), pp. 683-691.
49. Porter RM and Lane EB. Phenotypes, genotypes and their contribution to understanding keratin function. Trends Genet, 19 (2003), pp. 278-285.
50. Yaojiong Wu, Liwen Chen, Scott P, and Tredget and Edward E. Mesenchymal stem cells enhance wound healing through differentiation and angiogenesis. Stem Cells, 25 (2007), pp. 2648 -2265.
51. Michalczyk K and Ziman M. Nestin structure and predicted function in cellular cytoskeletal organisation. Histol. Histopathol, 20 (2005), pp. 665-671.
52. Morikuni Tobita, Hakan Orbay and Hiroshi Mizuno. Adipose-derived Stem Cells: Current Findings and Future Perspectives. Hopkins CME. 2011.
53. Zhao Q, Eberspaecher H, Lefebvre V and Crombrugghe B. Parallel expression of Sox9 and Col2a1 in cells undergoing chondrogenesis. Dev.Dyn. 209 (1999), pp. 377-386.
54. Hani Awad, Quinn Wickham, Holly Leddy, Jeffrey Gimble and Farshid Guilak. Chondrogenic differentiation of adipose-derived adult stem cells in agarose, alginate, and gelatin scaffolds. Biomaterials, 25 (2004), pp. 3211-3222.
55. Eileen Dawson, Gazell Mapili, Kathryn Erickson and Sabia Taqvi. Biomaterials for stem cell differentiation. Advanced Drug Delivery Reviews, 60 (2008), pp. 215-228.
56. Zuk PA, Zhu M, Ashjian P, Ugarte DA, Huang JI, Mizuno H and Hedrick MH. Human adipose tissue is a source of multipotent stem cells. Mol. Biol. Cell, 13 (2002), pp. 4279-4295.
57. Susan Liao, Casey Chan and Ramakrishna. Stem cells and biomimetic materials strategies for tissue engineering. Materials Science and Engineering C, 28 (2008), pp. 1189-1202.
58. Roman Salasznyk, William Williams, Adele Boskey and Anna Batorsky. Adhesion to vitronectin and collagen I promotes osteogenic differentiation of human mesenchymal stem cells. Biomed Biotechnol, 1 (2004), pp. 24-34.
59. Hoemann CD, Gabalawy HE and Kee MD. In vitro osteogenesis assays: Influence of the primary cell source on alkaline phosphatase activity and mineralization. Pathologie Biologie, 57 (2009), pp. 318-323.
60. Bettina Lindroos, Riitta Suuronen and Susanna Miettinen. The Potential of Adipose stem cells in regenerative medicine. Stem Cell Rev, 7 (2011), pp. 269-291.
61. Joseph Jagur-Grodzinski. Polymers for tissue engineering, medical devices, and regenerative medicine: Concise general review of recent studies. Polym. Adv. Technol, 17 (2006), pp. 395-418.
62. Conejero JA, Lee JA, Parrett BM, Terry M, Wear-Maggitti K and Grant RT. Repair of palatal bone defects using osteogenically differentiated fat-derived stem cells. Plast Reconstr Surg, 117 (2006), pp. 857-863.
63. Cowan CM, Shi Y, Aalami O and Chou YF. Adipose-derived adult stromal cells heal critical-size mouse calvarial defects. Nat Biotechnol, 22 (2004), pp. 560-567.
64. Haibin Zhou, Linshan Shang, Xiyu Zhang, Guimin Gao, Chenhong Guo, Bingxi Chen, Qiji Liu, Yaoqin Gong and Changshun Shao. Resveratrol augments the canonical Wnt signaling pathway in promoting osteoblastic differentiation of multipotent mesenchymal cells. Experimental cell research, 315 (2009), pp. 2 953-2962.
65. Jeon O, Rhie JW, Kwon IK and Kim JH. In vivo bone formation following transplantation of human adiposederived stromal cells that are not differentiated osteogenically. Tissue Eng Part A, 14 (2008), pp. 1285-1294.