Improvement of the Cell-loading Efficiency of Biomaterials by Inoculation with Stem Cell-based Microspheres,in Osteogenesis

Abstract

In critical-size bone defects, autologous or allogenic cells are required in addition to compatible biomaterials for the successful defect healing. State of the art inoculation methods of biomaterials are based on the application of cell suspensions to the biomaterial. However, only less amounts of cells can be applied and sufficient adhesion to the material is required. Therefore, it was investigated whether the advantages of stem cell-based microspheres and insoluble collagenous bone matrix (ICBM) scaffolds can be combined which can lead to an advancement in cell seeding on biomaterials. Microspheres were produced from unrestricted somatic stem cells from human umbilical cord blood and were mounted on ICBM scaffolds. Following the incubation with osteogenic or control medium, the constructs were analyzed histologically after 3, 7, 14, and 28 days. Alizarin Red S and von Kossa staining revealed microsphere mineralization after 3 days in osteogenic and after 14 days in control medium. Meanwhile, a time-dependent increase in tissue, growing out of the microspheres, was detected. Our results provide evidence that microsphere–ICBM constructs are promising candidates for approaches of bone regeneration. They allow the transfer of substantially high numbers of cells in partially mineralized constructs.

Keywords

biocompatibility bone tissue engineering scaffold stem cell mineralization

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References

McKee

M.D.

Nanci

. Osteopontin: An Interfacial Extracellular Matrix Protein in Mineralized Tissues, Connect. Tissue Res., 1996: 35: 197–205.

Walker

P.S.

Gold

B.L

. The Tribology (Friction, Lubrication and Wear) of All-metal Artificial Hip Joints 1971, Clin. Orthop. Relat. Res., 1996: S4–S10.

Kurashina

Kurita

Kotani

Takeuchi

Hirano

. In Vivo Study of a Calcium Phosphate Cement Consisting of Alpha-tricalcium Phosphate/Dicalcium Phosphate Dibasic/Tetracalcium Phosphate Monoxide, Biomaterials, 1997: 18: 147–151.

Lewis

. Properties of Acrylic Bone Cement: State of the Art Review, J. Biomed. Mater. Res., 1997: 38: 155–182.

Rokkanen

P.U

. Bioabsorbable Fixation Devices in Orthopaedics and Traumatology, Ann. Chir. Gynaecol., 1998: 87: 13–20.

Middleton

J.C.

Tipton

A.J

. Synthetic Biodegradable Polymers as Orthopedic Devices, Biomaterials, 2000: 21: 2335–2346.

Tuli

S.M.

Gupta

K.B

. Bridging of Large Chronic Osteoperiosteal Gaps by Allogeneic Decalcified Bone Matrix Implants in Rabbits, J. Trauma, 1981: 21: 894–898.

Handschel

Wiesmann

H.P.

Depprich

Kubler

N.R.

Meyer

. Cell-based Bone Reconstruction Therapies – Cell Sources, Int. J. Oral Maxillofac. Implants, 2006: 21: 890–898.

Yazar

Lin

C.H.

Wei

F.C

. One-stage Reconstruction of Composite Bone and Soft-tissue Defects in Traumatic Lower Extremities, Plast. Reconstr. Surg., 2004: 114: 1457–1466.

10.

Damien

C.J.

Parsons

J.R

. Bone Graft and Bone Graft Substitutes: A Review of Current Technology and Applications, J. Appl. Biomater., 1991: 2: 187–208.

11.

Meyer

Wiesmann

H.P.

Berr

Kubler

N.R.

Handschel

. Cell-based Bone Reconstruction Therapies – Principles of Clinical Approaches, Int. J. Oral Maxillofac. Implants, 2006: 21: 899–906.

12.

Hallab

N.J.

Bundy

K.J.

O'Connor

Clark

Moses

R.L

. Cell Adhesion to Biomaterials: Correlations Between Surface Charge, Surface Roughness, Adsorbed Protein, and Cell Morphology, J. Long Term Eff. Med. Implants, 1995: 5: 209–231.

13.

Depprich

Ommerborn

Zipprich

. Behavior of Osteoblastic Cells Cultured on Titanium and Structured Zirconia Surfaces, Head Face Med., 2008: 4: 29.

14.

Handschel

Berr

Depprich

. Compatibility of Embryonic Stem Cells with Biomaterials, J. Biomater. Appl., 2009: 23: 549–560.

15.

Naujoks

Langenbach

Berr

. Biocompatibility of Osteogenic Predifferentiated Human Cord Blood Stem Cells with Biomaterials and the Influence of the Biomaterial on the Process of Differentiation, J. Biomater. Appl., 2010 (Epub ahead of print).

16.

Handschel

Naujoks

Langenbach

. Comparison of Ectopic Bone Formation of Embryonic Stem Cells and Cord Blood Stem Cells In Vivo, Tissue Eng. Part A, 2010 (Epub ahead of print).

17.

Kögler

Sensken

Airey

J.A.

. A New Human Somatic Stem Cell from Placental Cord Blood with Intrinsic Pluripotent Differentiation Potential, J. Exp. Med., 2004: 200: 123–135.

18.

Kögler

. (2009). The Unrestricted Somatic Stem Cell (USSC) from Cord Blood for Regenerative Medicine, In: Meyer

Meyer

Handschel

Wiesmann

H.P.

(eds), Fundamentals of Tissue Engineering and Regenerative Medicine, Berlin, Heidelberg, Springer-Verlag, pp. 167–176.

19.

van den Berk

L.C.

Jansen

B.J.

Siebers-Vermeulen

K.G.

. Mesenchymal Stem Cells Respond to TNF but do not Produce TNF, J. Leukoc. Biol., 2010: 87: 281–289.

20.

Winter

Wang

X.N.

Daubener

. Suppression of Cellular Immunity by Cord Blood-derived Unrestricted Somatic Stem Cells is Cytokine Dependent, J. Cell Mol. Med., 2008: 13: 2465–2475.

21.

Handschel

J.G.

Depprich

R.A.

Kubler

N.R.

Wiesmann

H.P.

Ommerborn

Meyer

. Prospects of Micromass Culture Technology in Tissue Engineering, Head Face Med., 2007: 3: 4.

22.

Langenbach

Naujoks

Kersten-Thiele

P.V.

. Osteogenic Differentiation Influences Stem Cell Migration out of Scaffold Free Microspheres, Tissue Eng. Part A, 2009: 16: 759–766.

23.

Handschel

Berr

Depprich

R.A.

. Induction of Osteogenic Markers in Differentially Treated Cultures of Embryonic Stem Cells, Head Face Med., 2008: 4: 10.

24.

Aktas

Radke

T.F.

Strauer

B.E.

Wernet

Kogler

. Separation of Adult Bone Marrow Mononuclear Cells Using the Automated Closed Separation System Sepax, Cytotherapy, 2008: 10: 203–211.

25.

Bonewald

L.F.

Harris

S.E.

Rosser

. Von Kossa Staining Alone is not Sufficient to Confirm that Mineralization In Vitro Represents Bone Formation, Calcif. Tissue Int., 2003: 72: 537–547.

26.

Wang

Y.H

Liu

Maye

Rowe

D.W

. Examination of Mineralized Nodule Formation in Living Osteoblastic Cultures Using Fluorescent Dyes, Biotechnol. Prog., 2006: 22: 1697–1701.

27.

Wilson

C.E.

Dhert

W.J.

Van Blitterswijk

C.A.

Verbout

A.J.

De Bruijn

J.D

. Evaluating 3D Bone Tissue Engineered Constructs with Different Seeding Densities Using the AlamarBlue Assay and the Effect on In Vivo Bone Formation, J. Mater. Sci. Mater. Med., 2002: 13: 1265–1269.

28.

Holy

C.E.

Shoichet

M.S.

Davies

J.E

. Engineering Three-dimensional Bone Tissue In Vitro Using Biodegradable Scaffolds: Investigating Initial Cell-seeding Density and Culture Period, J. Biomed. Mater. Res., 2000: 51: 376–382.

29.

Bitar

Brown

R.A.

Salih

Kidane

A.G.

Knowles

J.C.

Nazhat

S.N

. Effect of Cell Density on Osteoblastic Differentiation and Matrix Degradation of Biomimetic Dense Collagen Scaffolds, Biomacromolecules, 2008: 9: 129–135.

30.

Hervy

Hoffman

Beckerle

M.C

. From the Membrane to the Nucleus and Back Again: Bifunctional Focal Adhesion Proteins, Curr. Opin. Cell Biol., 2006: 18: 524–532.

31.

Schwartz

M.A.

Assoian

R.K

. Integrins and Cell Proliferation: Regulation of Cyclin-dependent Kinases via Cytoplasmic Signaling Pathways, J. Cell. Sci., 2001: 114: 2553–2560.

32.

Kundu

A.K.

Khatiwala

C.B.

Putnam

A.J

. Extracellular Matrix Remodeling, Integrin Expression, and Downstream Signaling Pathways Influence the Osteogenic Differentiation of Mesenchymal Stem Cells on Poly(lactide-co-glycolide) Substrates, Tissue Eng. Part A, 2009: 15: 273–283.

33.

Xiao

Jiang

Thomas

. MAPK Pathways Activate and Phosphorylate the Osteoblast-specific Transcription Factor, Cbfa1, J. Biol. Chem., 2000: 275: 4453–4459.

34.

Franceschi

R.T.

Xiao

. Regulation of the Osteoblast-specific Transcription Factor, Runx2: Responsiveness to Multiple Signal Transduction Pathways, J. Cell Biochem., 2003: 88: 446–454.

35.

Bradshaw

A.D.

Graves

D.C.

Motamed

Sage

E.H

. SPARC-null Mice Exhibit Increased Adiposity Without Significant Differences in Overall Body Weight, Proc. Natl. Acad. Sci. U.S.A., 2003: 100: 6045–6050.

36.

Francki

Bradshaw

A.D.

Bassuk

J.A.

Howe

C.C.

Couser

W.G.

Sage

E.H

. SPARC Regulates the Expression of Collagen Type I and Transforming Growth Factor-beta1 in Mesangial Cells, J. Biol. Chem., 1999: 274: 32145–32152.

37.

Iruela-Arispe

M.L.

Vernon

R.B.

Jaenisch

Sage

E.H

. Type I Collagen-deficient Mov-13 Mice do not Retain SPARC in the Extracellular Matrix: Implications for Fibroblast Function, Dev. Dyn., 1996: 207: 171–183.