Stem Cells Dev. 2013 May 15;22(10):1588-601.

Enhanced Osteoblastogenesis of Adipose-Derived Stem Cells on Spermine Delivery via β-Catenin Activation

Serena Guidotti,1,2 Annalisa Facchini,2,3 Daniela Platano,1,2 Eleonora Olivotto,1,4 Manuela Minguzzi,1,2

Giovanni Trisolino,5 Giuseppe Filardo,6 Silvia Cetrullo,3 Benedetta Tantini,3 Ermanno Martucci,5

Andrea Facchini,1,2,4 Flavio Flamigni,3 and Rosa Maria Borzı`1,4

1 Laboratorio di Immunoreumatologia e Rigenerazione Tessutale, Istituto Ortopedico Rizzoli, Bologna, Italy.

2 Dipartimento di Medicina Clinica, Universita` di Bologna, Bologna, Italy.

3 Dipartimento di Biochimica, Universita` di Bologna, Bologna, Italy.

4 Dipartimento RIT, Laboratorio RAMSES, Istituto Ortopedico Rizzoli, Bologna, Italy.

5 Chirurgia ricostruttiva articolare dell’anca e del ginocchio, Istituto Ortopedico Rizzoli, Bologna, Italy.

6 Laboratorio di Biomeccanica e Innovazione Tecnologica, Clinica III, Istituto Ortopedico Rizzoli, Bologna, Italy.



The molecular mechanisms underlying spermine osteo-inductive activity on human adipose-derived stem cells (ASCs) grown in 3-dimensional (3D) cultures were investigated. Spermine belongs to the polyamine family, naturally occurring, positively charged polycations that are able to control several cellular processes. Spermine was used at a concentration close to that found in platelet-rich plasma (PRP), an autologous blood product containing growth and differentiation factors, which has recently become popular in in vitro and in vivo bone healing and engineering. Adipose tissue was surgically obtained from the hypodermis of patients undergoing hip arthroplasty. Patient age negatively affected both ASC yield and ASC ability to form 3D constructs. ASC 3D cultures were seeded in either non differentiating or chondrogenic conditions, with or without the addition of 5µM spermine to evaluate its osteogenic potential across 1, 2, and 3 weeks of maturation. Osteogenic medium was used as a reference. The effects of the addition of spermine on molecular markers of osteogenesis, at both gene and protein level, and mineralization were evaluated. The effects of spermine were temporally defined and responsible for the progression from the early to the mature osteoblast differentiation phases. Spermine initially promoted gene and protein expression of Runx-2, an early marker of the osteoblast lineage; then, it increased β-catenin expression and activation, which led to the induction of Osterix gene expression, the mature osteoblast commitment factor. The finding that spermine induces ASC to differentiate toward mature osteoblasts supports the use of these easily accessible mesenchymal stem cells coupled with PRP for orthopedic applications




Along with the expanding knowledge of cell  biology and the development of smart biomimetic scaffold, the use of stem cells opens interesting avenues in musculoskeletal regenerative medicine. Compared to bone marrow stromal cells (MSC) stem cells derived from adipose tissue (ASC) entered the scene of  regenerative medicine with some years of delay but offer interesting advances including the ease and low morbidity of their recovery and the higher precursor frequency per unit of processed tissue. Stem cells in orthopaedic regenerative medicine act both via an “immunomodulatory activity”  and a “plastic activity”, as precursors of cells of cartilage (chondrocytes) or bone (osteoblasts). Regarding their use as precursors of differentiated cells to heal bone or cartilage defects,  it is worthy to underline  the worldwide increasing clinical use  of”platelet rich plasma” or PRP, a safe and cheap blood derivative recovered from patient’s bedside through a fast concentration procedure which essentially increases the number of platelets per unit volume. According to distinct protocols used by different groups, PRP may also present a significant enrichment of leukocytes, as it happens with the PRP prepared following the procedure in use at the Rizzoli Orthopaedic Institute (Bologna, Italy).  Besides being of help in immobilizing stem cells, PRP  is a rich source of bioactive peptides and growth factors including those already known for their ability to increase proliferation of stem cells in vitro (Sanchez-Gonzalez et al., 2012).  But since in most clinical uses PRP is conserved aliquoted and freezed, ready to be delivered to patients in longitudinal follow up, we thought that its clinical efficacy could be at least partially dependent on something which became available when frozen blood cells are thawed and partially release their content. We in particular looked at the PRP content of polyamines (putrescine, spermidine and spermine), a family of molecules that exert a number of different cellular activities which globally control gene transcription at both DNA (gene demethylation) and RNA (RNA stability and translation) level, as well as protein posttranslational modifications as reviewed in  (Borzi et al., 2013). Through these pleiotropic activities polyamines are able to control cell growth and proliferation, apoptosis and autophagy, which need to be tightly regulated in a time and space ordered manner during stem cell differentiation and development. Indeed many old studies have pointed that stem cell condensation, the very first step in skeletal development, is accompanied by a dramatic increase in ornithine decarboxylase the pivotal polyamine biosynthetic enzyme. Furthermore, recent findings point at a strong anti-oxidant activity of  spermine which acts as a shield to protect DNA from hydrogen peroxide-induced oxidative damage (Korhonen et al., 2001). In keeping with this protective activity against oxidative damage,  a higher spermine content in the blood of healthy non-centenarians has been reported  (Pucciarelli et al., 2012).  Collectively, these findings suggest that polyamines can both fulfill a differentiating and protective role in regenerative medicine.

We found that PRP prepared according to the Rizzoli procedure indeed contains polyamines in the µM range and that spermine addition at the concentration found in PRP, increases ASC differentiation along the osteoblast lineage and at the same time protects the cells from oxidative DNA damage. These findings have been collected with ASC cultured in 3-D, to more closely approach the in vivo settings. In this perspective, polyamine delivery can increase the success of regenerative medicine approaches in those conditions (age, obesity) that are associated with a systemic oxidative stress, responsible for stem cell exhaustion. The differentiative ability of spermine is exerted through the enhancement of expression and nuclear translocation of β-catenin and induction of osterix that represents the no-return step from the early (from stem cells to osteo-chondro precursors) to the late (from the osteo-chondro precursor to the mature osteoblasts) osteoblast differentiative phases. Spermine addition proved to reduce the level of apoptosis as measured by the staining of active effector caspase 3, and this resulted in an increased number of  viable cells surviving the apoptotic pressure associated with the process of asymmetric division ongoing in the differentiating 3-D constructs.

Our findings provide molecular explanation for the rationale of using prolotherapy approaches of combined ASC+PRP for treating focal bone defects.



Borzi RM, Guidotti S, Minguzzi M, Facchini A, Platano D, Trisolino G, Filardo G, Cetrullo S, D’Adamo S, Stefanelli C, Flamigni F. 2013. Polyamine delivery as a tool to modulate stem cell differentiation in skeletal tissue engineering. Amino Acids. Nov 19. [Epub ahead of print]

Korhonen VP, Niiranen K, Halmekyto M, Pietila M, Diegelman P, Parkkinen JJ, Eloranta T, Porter CW, Alhonen L, Janne J. 2001. Spermine deficiency resulting from targeted disruption of the spermine synthase gene in embryonic stem cells leads to enhanced sensitivity to antiproliferative drugs. Mol Pharmacol 59(2):231-238.

Pucciarelli S, Moreschini B, Micozzi D, De Fronzo GS, Carpi FM, Polzonetti V, Vincenzetti S, Mignini F, Napolioni V. 2012. Spermidine and spermine are enriched in whole blood of nona/centenarians. Rejuvenation Res 15(6):590-595.

Sanchez-Gonzalez DJ, Mendez-Bolaina E, Trejo-Bahena NI. 2012. Platelet-rich plasma peptides: key for regeneration. Int J Pept 2012:532519.


Figure1Figure 1. Spermine activities in  osteoblastogenesis of adipose derived stem cells.

Along each step of the differentiation pathways, transcription factors with an inducing effect are indicated in red, while those with an inhibiting effect are indicated in green.

The upper portion of the figure outlines the experimental plan.  Adipose tissue samples derived from the surgical subcutaneous area were minced and treated with enzymatic digestion and centrifugation. The pellet corresponding to the “stromal vascular fraction” was plated in order to select the adherent cells, and to isolate the “adipose tissue derived mesenchymal stem cells” (ASC).  After flow cytometric characterization of their phenotype, ASC were differentiated in presence of spermine in 3-dimensional culture condition to recapitulate mesenchymal cell condensation, the very first step in skeletal development.

The central portion of the figure outlines the alternative differentiation pathways leading to osteoblastogenesis and bone formation. Spermine addition prompted ASC in 3-D culture to enter the osteoblastogenesis pathway that mimics “intramembranous ossification” rather than “chondrogenesis followed by endochondral ossification”. This in vitro osteoblastogenesis was sustained by spermine-dependent increased expression and activation of  pivotal differentiation driving transcription factors (β-catenin, Runx2, Osterix).

The lower portion of the figure lists the known biological processes affected by spermine flanked by the spermine dependent molecular effects described in our manuscript.


Acknowledgements:  This work was supported by FIRB (MIUR, Italy) RBAP10KCNS and Fondi cinque per mille (Ministero della Salute, Italy).


Rosa Maria Borzì, MD.

Istituto Ortopedico Rizzoli
Via di Barbiano 1/10

40136, Bologna

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