www.elsevier.com/locate/lifescie

Life Sciences 73 (2003) 1973–1983

Polydeoxyribonucleotide (PDRN) promotes human osteoblast

proliferation: A new proposal for bone tissue repair

Stefano Guizzardia,*, Carlo Gallia, Paolo Govonia, Renata Borattob, Giulia Cattarinic,Desiree Martinid, Silvana Bellettia, Renato Scandroglioa

aDepartment of Experimental Medicine - Section of Histology, Via Volturno 39, University of Parma, 43100 Parma - ItalybDepartment of Experimental Medicine - Section of Anatomy, Strada Nuova 65, University of Pavia, Pavia - Italy

cMastelli Laboratory, Via Bussana Vecchia 32, San Remo (IM) - Italyd Institute of Human Anatomy, Via Irnerio 48, University of Bologna, Bologna - Italy

Received 11 June 2002; accepted 4 April 2003

Abstract

Several researchers have recently shed new light upon the importance of extracellular nucleotides and

nucleosides to stimulate cells growth. PDRN, a mixture of deoxyribonucleotides polymers of different lengths,

has recently demonstrated to stimulate ‘‘in vitro’’ fibroblast proliferation and collagen production, probably

stimulating the purinergic receptor system. In this work we evaluated the effects of PDRN on human cultured

osteoblasts, focusing our attention on cell proliferation and alkaline phosphatase activity. PDRN at a con-

centration of 100Ag/ml induce an increase in osteoblasts growth after 6 days as compared to control ( + 21%).

The addition of DMPX 50 AM and suramine (P2 inhibitor) 10 AM give different results: suramine has no

significant effect, while DPMX reduce, even if partially, the PDRN induced cell growth. The alkaline

phosphatase activity shows a gradual enhancement starting from day 0 to day 10, even if PDRN treated cells,

examined at day 6, present a sensibly lower phosphatase activity when compared to controls. Our data

demonstrate that PDRN acts as an osteoblast growth stimulator. Its action is partially due to a stimulation of the

purinergic system mediated by A2 purinoreceptors, however we can not exclude the involvement of other

mechanism like salvage pathway.

D 2003 Elsevier Inc. All rights reserved.

Keywords: Polydeoxyribonucleotide; Osteoblast; Purinergic receptor; DMPX

0024-3205/03/$ - see front matter D 2003 Elsevier Inc. All rights reserved.

doi:10.1016/S0024-3205(03)00547-2

* Corresponding author. Tel.: +39521903911; fax: +39521903912.

E-mail address: stegui@unipr.it (S. Guizzardi).

S. Guizzardi et al. / Life Sciences 73 (2003) 1973–19831974

Introduction

Modern surgical techniques in orthopaedic and maxillo-facial surgery always request a fast bone

tissue regeneration to fill gaps created by surgery or rapidly increase fixation of bones implants.

Currently, the autogenous bone graft is the most used device, a kind of golden standard (Lorenzetti et al.,

1998; Jensen et al., 1998), mainly for two reasons; it is a sort of structure which can be integrated into

the receiving site, thus filling the existing gap, but it also provides an osteoinductive signal for the

mesenchymal precursor cells, that are induced to proliferate and to differentiate into active osteoblasts,

thereby accelerating the healing process (Lane et al., 1999). Thus far, bone is the only true

osteoinductive material able to promote and induce new bone tissue formation from osteoblast

precursors, stimulating stem cell differentiation into mature osteoblasts.

The osteoinductive properties of natural bone are due to the presence of Bone Morphogenetic

Proteins (BMP) in the matrix (Harakas, 1984; Reddi, 1992; Urist, 1965; Service, 2000). This protein

has been first isolated and then produced as a cloned series of molecules from the BMP protein

family (Urist and Strates, 1971; Service, 2000). Above all Bmp-2 and Bmp-7 are extremely powerful

mitogenic and differentiating agents (Boden, 1999). However their effect is not as long standing as

wished and, in order to work properly, they have to be utilized at very high, non-physiological doses

(even 6 orders of magnitude higher) whose long-term effects have not been thoroughly yet

understood (Service, 2000). Growth factors, like PDGF, FGF, IGF, and TGF-h are important

molecules implicated in the bone regeneration processes (Canalis et al., 1988, Joyce et al., 1990,

1991; Bourque et al., 1993; Einhorn, 1996) and are currently under examination as therapeutic

agents. Their use, already largely experimented on rodents has proven to be difficult to translate to

humans, and there are still many doubts about the proper doses that should be used (Bruder et al.,

1999).

Several researchers have recently shed new light on the importance of the action of extracellular

nucleotides and nucleosides to increase proliferation and activity of cells of different tissues. Nucleic

acids, nucleosides deriving from their fragmentation, and nucleotides physiologically diffuse into the

extracellular environment as a result of cell lysis (Born and Kratzer, 1984; Bowler et al., 2001)

following cellular death, possibly providing a local stimulus for tissue regeneration. It has been

demonstrated that they act as growth promoters for fibroblasts (Muratore et al., 1997; Sini et al., 1999),

osteoblasts (Nakamura et al., 2000; Bowler et al., 1999), endothelial cells, and neuroglia (Rathbone et

al., 1992a,b,c). Similar results have been obtained in in vivo experimental models on rats (Montesinos

et al., 2002).

They also act in synergy with different growth factors like PDGF, FGF, EGF (Ding Gji et al., 1990;

Chavan and Haley, 1994), modulate cytokines and growth factor production and even influence

immunologic responses (Edington, 1992; Adjej et al., 1995; Gailit and Clarck, 1994).

Furthermore, there is evidence that polydeoxyribonucleotides offer a protective and regenerative

effect on UV irradiated cell cultures and on lethally exposed rats (Musk et al., 1989; Henning et al.,

1996).

Nucleotides like ATP can also be released by cell membranes (Bowler et al., 2001), thereby

maintaining a threshold level which is probably highly relevant as an autocrine-paracrine message in

the modulation of signal transduction pathways, even in the definition of the basal cellular activity

(Ostrom et al., 2000). Nucleotides and nucleosides are supposed to act in different ways: first they

can stimulate nucleic acid synthesis through the salvage pathways (Viassov et al., 1994; Nakamura

S. Guizzardi et al. / Life Sciences 73 (2003) 1973–1983 1975

et al., 2000), and their effect is mediated by binding to the purinergic receptors (Bianchini et al.,

1981; Henning et al., 1996; Bowler et al., 1999; 2001; Thellung et al., 1999; Nakamura et al.,

2000).

These receptors can be divided into two main classes: P1 receptors, responsive to nucleosides, and P2

receptors, which bind nucleotides, more specifically distinguished not only by their different binding

affinity, but also by their signal transduction mechanisms. P2x are well known for their capacity to

trigger a proliferative response in osteoblasts, under ATP stimulation (Rathbone et al., 1992a,b,c; Bowler

et al., 1999), whereas P2y receptors have been recently proved to strengthen the action of PTH on bone

cell cultures (Henning et al., 1996).

Similarly, S. Thellung et al. (1999) have shown that, when stimulated by a variety of nucleosides,

A2 receptors (a P1 subclass) induce proliferation of cultured fibroblasts. Similar in vitro and in vivo

studies have demonstrated the role of A2 receptors in wound healing process (Vega et al., 2002).

Polydeoxyribonucleotide (PDRN) is a compound holding a mixture of deoxyribonucleotide

polymers of different lengths, comprised between 50 and 2000 base pairs, together with nucleosides

obtained from salmon trout (Oncorhynchus mykiss) sperm by a purification and sterilization process

that guarantees a high percentage of DNA and the absence of active proteins or peptides (Tonello et

al., 1996). This compound has been used to stimulate healing in X ray induced tissue lesions

(Bianchini et al., 1981; Bigliardi, 1982). It has been supposed that PDRN is cleaved by active cell

membrane enzymes, providing a source for deoxynucleotides and deoxynucleosides (Nakamura et al.,

2000).

This paper evaluates the effects of PDRN on human osteoblasts cultures, focusing on cell pro-

liferation and alkaline phosphatase activity. Moreover we have studied the role of purinergic inhibitors

on PDRN activity.

Methods

Primary explants culture and secondary culture

Human osteoblasts have been obtained from jawbone specimens resulting from a surgical

intervention on a 5-year-old patient, and isolated as previously described (Beresford et al., 1984;

Siggelkow et al., 1998). Briefly, bone fragments were cleaned from present soft tissues remnants,

thoroughly rinsed in phosphate buffered saline (PBS, Sigma Chemicals, St. Louis MO, USA) and

then maintained in DMEM (Bio Whittaker, Walkersville, USA) with 10% FBS (Foetal Bovine

Serum, Bio Whittaker), glutamine 4 mM, streptomycin (100 Ag/ml) and penicillin (100 U/ml)

(Penstrep - GibcoBRL, Life Technologies B.V. Breda, The Netherlands) at 37 jC in modified

atmosphere (5% CO2 95% air) until cell outgrowths appeared. Once confluent, cells were detached

by trypsin (trypsin-EDTA 0,25%) (Sigma) seeded in Petri dishes (Falcon, Becton Dickinson Europe,

Meylan, France) and feeded three times a week with DMEM complete medium, enriched with fresh

ascorbic acid 250 AM (Gallagher et al., 1996) to maintain a differentiated osteoblastic phenotype.

Cell populations obtained was further characterized by the evaluation of alkaline phosphatase activity

according to Farley et al. (Farley et al., 1994) and by osteocalcin production using the Osteocalcin

ELISA Kit (DAKO s.p.a, Milano, Italy) using a normal skin fibroblast population as negative

control.

Assessment of cell growth

The effect of PDRN stimulation on osteoblast growth was assessed by directly counting the cell

population. Cells from the second to the fourth in vitro passage, were seeded at 15000–20000 cells/ cm2

onto 24-well plates (Falcon) in DMEM plus ascorbic acid 250 Am.

Twenty-four hours after seeding, cells were treated for 2, 4, and 6 days with PDRN 100Ag/ml,

(Mastelli, Sanremo, Italy) in DMEM plus FBS at different concentrations (1%, 5%, 10%).

The number of cells was evaluated for each sample after 0 (control), 2, 4, and 6 days of

treatment by detaching and direct counting with a Coulter Counter (Coulter Electronics Limited,

England). Briefly, each sample was washed with PBS (Ca++ and Mg+ + free), detached with 300 Alof trypsin-EDTA and transferred into cold physiological solution for counting.

In another set of experiments, the rate of growth inhibition during treatment with PDRN was also

measured after addition of DMPX (an A2 receptor inhibitor) and Suramine (a specific P2 inhibitor) to

the medium.

For this experiment human osteoblasts were seeded onto four 24-wells plates, at a density of 20000/

cm2, in DMEM plus FBS 10% and 250 Am ascorbic acid. Twenty-four hours after seeding cells were

treated for 2, 4, 6 days with PDRN 100 Ag/ml plus DMPX 50 AM (Thellung et al., 1999) or plus

Suramine 10AM (Nakamura et al., 2000) in DMEM supplemented with 10% FBS and compared with a

population treated only with PDRN 100 Ag/ml. Cell samples were then counted at 0, 2, 4, 6 days of

treatment as previously described.

Assay for detecting alkaline phosphatase activity

The assay for alkaline phosphatase activity was carried out according to Farley et al. (1994).

For this purpose cells from the second to the fourth in vitro passages were seeded onto 12-wells

plates at a density of 15000–20000 cells/cm2 in DMEM containing 10% FBS. Twenty-four hours

after seeding cells were treated with PDRN 100Ag/ml for 2, 4, 6, 8 and 10 days. Determination

of ALP activity was performed as follow: at the end of treatment cells were washed with PBS

then lysed in Triton 0.1% (Triton X-100) in PBS then frozen at � 20 jC and thawed. 100Al of

cell lysates were mixed with 200Al of 10 mM p-Nitrophenyl Phospate and 100Al of 1.5 M 2-

amino-2-methyl-1-propanol buffer (AMP). Samples were then incubated for 1 h at 37 jC. ALP

activity was measured for each sample by absorbance reading at 400 nm with the Helios

spectrophotometer (Spectronic Unicam, Cambridge, UK) and corrected for cell number determined

in parallel.

Chemicals

Ascorbic acid, DMPX (Dimethyl-propargyl-xanthine), Suramine, p-Nitrophenyl Phospate, 2-amino-

2-methyl-1-propanol (AMP) buffer and all other chemicals or reagent, if not otherwise indicated, were

from SIGMA Chemicals (St. Louis MO, USA).

Statistical Analysis

All the experiments were repeated three times with three determinations for each sample.

S. Guizzardi et al. / Life Sciences 73 (2003) 1973–19831976

S. Guizzardi et al. / Life Sciences 73 (2003) 1973–1983 1977

For the statistical analysis, the collected data were analysed by Anova for Repeated Measure (Split-

Plot Design) after square-root transformation (frequency count of cells) and Bonferroni’s multiple

comparison test. All the probabilistic descriptive procedures were carried out on a Power Macintosh G3

computer, using SPSS programs for SPSS International B.V.

Results

Cell growth stimulation

Osteoblasts treated with PDRN at different FBS concentrations exhibited different behaviour (Fig. 1).

The presence of PDRN at the lowest concentration of FBS (1%) used in this study, had no significant

effect on cell growth until the fourth day of treatment.

After two days the difference between PDRN treated and untreated cells was not relevant, and

only starting from 4th day the difference between the two groups reached a significance (P <

0.05). At FBS concentration of 5% the presence of PDRN caused a more pronounced difference;

just after 2 days the treated cultures showed an increased cell growth (P < 0.05) when compared

to the control, and this difference was also maintained after 6 days. The best growth rate was

obtained after treatment with PDRN at highest FBS concentration (10%); in this condition the

addition of PDRN determined just after 2 days a statistically significant increased cell growth (P <

0.01) that reached its maximum after 6 days (P < 0.001) with an increase of cell number of

about 21%.

Inhibitors effect

The experiments were conducted at a concentration of 50 AM DMPX, as suggested by previous

authors (Thellung et al., 1999).

Fig. 1. PDRN stimulation of human osteoblasts cell growth at different serum concentration. Cell proliferation was assessed

in presence of increasing FBS concentrations (1%,5%,10%) by direct counting of cell population (see Material and Methods)

after 2,4,6 days in presence or not of 100 Ag PDRN. Data represents means of four independent determination F SEM

indicated (*p < 0.05; **p < 0.01; ***p < 0.001).

Fig. 2. Effect of DMPX on PDRN stimulation of human osteoblasts cell growth. Cell proliferation in presence of 100 AgPDRN +/- 50 AM DMPX was assessed by direct counting of cell population (see Material and Methods) after 2,4,6 days.

Data represents means of four independent determination F SEM.

S. Guizzardi et al. / Life Sciences 73 (2003) 1973–19831978

The effect of 50 AM DMPX inhibitor on PDRN treated cells is shown in Fig. 2. PDRN activity was

inhibited after 48 hrs. of treatment and was still evident on day six; nevertheless at this time, DMPX

treated cells presented only a reduction of 42,9% in comparison to PDRN treated samples.

The difference between the groups (PDRN only vs. PDRN + DMPX treated cells) was significant

after 4 and 6 days of treatment (p < 0.001).

The effect of Suramine inhibitor on treated cells is shown in Fig. 3. The addition of this inhibitor at 10

AM had no significant effect on PDRN-induced cell growth.

Fig. 3. Effect of Suramine on PDRN stimulation of human osteoblasts cell growth. Cell proliferation in presence of 100 AgPDRN +/- suramine at different concentrations was assessed by direct counting of cell population (see Material and Methods)

after 2,4,6 days. Data represents means of four independent determination F SEM.

Fig. 4. Human osteoblasts ALP activity during PDRN treatment. Specific alkaline phosphatase activity of human osteoblasts

was measured (see Material and Methods) after 6 and 10 days of treatment with 100 Ag PDRN. Data represents means of four

independent determination F SEM.

S. Guizzardi et al. / Life Sciences 73 (2003) 1973–1983 1979

Moreover the control (no treatments) cell population mean value always appeared lower when

compared with PDRN alone and with PDRN plus Suramine 10 AM on the sixth day.

The growth rate of PDRN and PDRN plus inhibitors showed an increase until the fourth day (p <

0.001) while only with Suramine 10 AM we observed an increase on the sixth day.

Higher Suramine concentrations determined a strong reduction in cell number also in comparison to

control just after 48 hrs.

Alkaline Phosphatase

The alkaline phosphatase activity shows a gradual increase starting from day 0 to day 10, in both

groups with some differences between PDRN treated and control on intermediate days. It was easy to

note a slight difference in the enzyme activity rate between control and treated samples on day 6, while

there was not significant discrepancy between the two groups on day 10. The specific enzyme activity

related to cell number maintains a constant course through time, even if the assay shows a steady

increment in the levels of enzyme activity from day 0 to day 10. As far as the differences between the

studied groups are concerned, the PDRN treated cells examined at day 6, present a significant lower

phosphatase activity when compared with controls (P < 0.01), as shown in Fig. 4.

Discussion

The availability in bone surgery of molecules whose effect is to stimulate osteoblast growth is an

argument of great interest in bone surgery. In particular osteoblast stimulators can be useful in situations

in which a bone defect must be filled with biomaterials, inducing in the host osteoblast a more rapid

proliferation, new bone formation, and finally a reduction in healing time. The present study examines

the capacity of PDRN, a mixture of deoxyribonucleotides polymers of different length, to induce growth

and enhance activity of cultured human osteoblast. In vitro PDRN effects has been previously

investigated on cultured fibroblast (Sini et al., 1999; Nakamura et al., 2000) demonstrating the

S. Guizzardi et al. / Life Sciences 73 (2003) 1973–19831980

effectiveness on proliferation and collagen production. Other studies have demonstrated that the effects

of PDRN on fibroblast are mediated by purinergic receptors and in particular the A2 subtype (Thellung

et al., 1999).

We have analysed the effect of PDRN on osteoblast growth and alkaline phosphatase production, and

have also investigated if the mechanisms of cell stimulation are similar to those observed or

hypothesised for other cell types.

In the first part of our experimental protocol we observed that FBS concentration is a factor that

strongly influence the osteoblast cell growth in presence of PDRN. Osteoblasts treated with PDRN

exhibit an optimal growth rate depending on FBS concentration, and the best results are reached with

FBS 10% (P < 0.001). On the other side in FBS deprived cultures no effect of PDRN is detectable

(data not shown). This initial finding strongly suggests the importance of FBS on PDRN induced

osteoblast growth. This effect could be explained by the presence of some enzymes active on

oligonucleotides, which acting through a depolymerization process, produce free purine nucleotides

and nucleosides that could bind to purinergic receptors (Nakamura et al., 2000). The addition of PDRN

(100Ag/ml) to the medium caused an increase in growth just 48 hrs after; this increase reached its

maximum after 6 days and then decreased when the cultures became confluent. Our findings of an

increased cell growth of about 21% after 6 days are in agreement with those observed by Sini et al.

(1999), which have shown that the same PDRN concentrations determined an increased fibroblast

growth of about 20%.

This result has been implemented by the analysis of the curves obtained after treatment with

DMPX, (A2 purinergic receptor inhibitor) and Suramine, (P2 purinergic receptor inhibitors), that

offer a contribution for a better understanding of PDRN effects on cultured cells. DMPX induces a

partial inhibition of PDRN-induced cell growth (� 42.9%), while Suramine 10 AM has no

significant effect; on the opposite, higher concentrations of this molecule seem to exert a inhibitory

effect on cell growth, even in comparison to controls, probably implying different mechanisms of

action. These data suggests the involvement, even if partial, of A2 receptors in the oligonucleotides

stimulated growth; PDRN, once in contact with cells, is probably degraded into simpler components

like nucleosides that stimulate the A2 adenosine receptors. Nevertheless the PDRN growth is not

completely inhibited, and it suggest that the Adenosine A2 receptor is not the exclusive mechanism

of PDRN action.

Finally, the analysis of the alkaline phosphatase activity after stimulation with PDRN does not show a

linear correspondence between cell growth and ALP activity; its slower increase in the treated samples

has been evidenced, whereas after 10 days the activity was similar for treated and untreated cells. These

data, in apparent contrast with the growth curves, can be explained by the fact that alkaline phosphatase

production is not constant during the whole cell cycle, but it is produced only in the latest G phase prior

to M phase (Fedarko et al., 1990). Furthermore a probably reduction of the cell cycle length as

consequence of increased proliferation determined a shortening of the G phase. On the other side some

authors report that when cultured osteoblasts are stimulated to proliferate with agents that promote cell

division (growth factors and hormones) their phosphatase activity dropped (Chin, 1984). Interestingly

when the cultures become confluent and the cell growth increase stops, treated and untreated samples

show similar activity.

From the analysis of the collected data, we can conclude that PDRN acts as osteoblast growth

stimulator; its action is partially due to a stimulation of the purinergic system mediated by A2

purinoreceptors, but we can not exclude the involvement of other mechanism like salvage pathway

S. Guizzardi et al. / Life Sciences 73 (2003) 1973–1983 1981

(Nakamura et al., 2000). For these reasons PDRN may be a useful osteoblast stimulator, and play an

important role in repairing bone defects.

Acknowledgements

This work was supported by a grant FIL, University of Parma. Thank to Mastelli s.r.l for the

assistance on prepare and purify PDRN.

References

Adjej, A.A., Yamamoto, S., Kulkarni, A., 1995. Nucleic acid and /or their components: a possible role in immune function.

Journal of Nutritional Science and Vitaminology 41, 1–16.

Beresford, J.N., Gallagher, J.A., Poser, J.W., Russel, R.G.G., 1984. Production of osteocalcin by human bone cells in vitro.

Effects of 1,25-(OH)2D3 24,25-(OH)2D3, parathyroid hormone, and glucocorticoids. Metabolic Bone Disease & Related

Research 5, 229–234.

Bianchini, P., Tellini, N., Morani, A.M., Follano, M.G., 1981. Pharmacological data on polydeoxyribonucleotide of human

placenta. International Journal of Tissue Reactions 3, 151–154.

Bigliardi, P., 1982. Treatment of acute radiodermatitis of first and second degrees with semi-greasy placenta ointment. Interna-

tional Journal of Tissue Reactions 4, 153–154.

Boden, S.D., 1999. Bioactive factors for bone tissue engineering. Clinical Orthopaedics and Related Research 367S, 84–94.

Born, G.V., Kratzer, M.A., 1984. Source and concentration of extracellular adenosine triphosphate during haemostasis in rats,

rabbits and man. Journal Physiology (London) 354, 419–429.

Bourque, W.T., Gross, M., Hall, B.K., 1993. Expression of four growth factors during fracture repair. The International Journal

of Developmental Biology 37, 573–579.

Bowler, W.B., Buckley, K.A., Gartland, A., Hipskind, R.A., Bilbe, G., Gallagher, J.A., 2001. Extracellular Nucleotide

signaling: a mechanism for integrating local and systemic responses in the activation of bone remodeling. Bone 28 (5),

507–512.

Bowler, W.B., Dixon, C.J., Halleux, C., Maier, R., Bilbe, G., Fraser, W.D., Gallagher, J.A., Hipskind, R.A., 1999. Signaling in

human osteoblasts by extracellular nucleotides. Their weak induction of the c-fos oncogene via Ca2+ mobilization is

strongly potentiated by a parathyroid hormone/cAMP-dependent protein kinase pathway indipendently of mitogen-activated

protein kinase. Journal of Biological Chemistry. 274, 14315–14324.

Bruder, S.P., Fox, B.S., 1999. Tissue engineering of bone. Clinical Orthopaedic and Related Research 367S, S68–S83.

Canalis, E., McCarthy, T., Centerella, M., 1988. Growth factors and the regulation of bone remodeling. Journal of Clinical

Investigation 81, 277–281.

Chavan, A.J., Haley, B.E., 1994. Interaction of nucleotides with acidic fibroblast growth factor (FGF-1). Biochemistry 33,

193–7202.

Chin, J.E., 1984. Production and characterization of matrix vesicles by chicken epiphiseal growth plate chondrocytes in primary

culture and regulation by physiological factors. Ph.D. Thesis, University of South, 166–168.

Ding Gji, W., Ning-Na, H., Heppel, L.A., 1990. Extracellular ATP shows synergistic enhancement of DNA synthesis when

combined with agents that are active in wound healing or as neurotransmitters. Biochemistry Biophysic Resesearch

Communications 166, 251–258.

Edington, S.M., 1992. DNA and RNA therapeutics: unsolved riddles. Bio Technology 10, 993–996.

Einhorn, T.A., 1996. Enhancement of fracture healing. Instructional Course Lectures 45, 401–416.

Farley, J.R., Hall, S.L., Tanner, M.A., Wergedal, J.E., 1994. Specific activity of scheletal alkaline phosphatase in human

osteoblast-line cells regulated by phosphate, phosphate esters, and phosphate analogs and release of alkaline phosphatase

activity inversely regulated by calcium. Journal of Bone Mineral Research 9 (4), 497–508.

Fedarko, N.S., Bianco, P., Vetter, U., Gheron Robey, P., 1990. Human bone celle enzyme expression and cellular heterogeneity:

correlation of alkaline phosphatase enzyme activity with cell cycle. Journal of Cellular Phisiology 144, 115–121.

S. Guizzardi et al. / Life Sciences 73 (2003) 1973–19831982

Gailit, J., Clarck, A.F., 1994. Wound repair in the context of extracellular matrix. Current Opinion in Cell Biology 6,

717–725.

Gallagher, J.A., Gundle, R., Beresford, J., 1996. Isolation and culture of bone-forming cells (osteoblasts) from human bone. In:

Jones, G.E. (Ed.), Human Cell Culture Protocols. Humana Press, Totowa NJ, pp. 232–257.

Harakas, N.K., 1984. Demineralized bone matrix-induced osteogenesis. Clinical Orthopaedic and Related Research 188,

239–251.

Henning, U.G., Wang, Q., Gee, N.H., Borstel, R.C., 1996. Protection and repair of gamma radiation induced lesions in mice

with DNA or deoxyribonucleotides treatments. Mutatation Research 35, 25–30.

Jensen, O.T., Shulman, L.B., Block, M.S., Iacono, V.J., 1998. Report of the sinus consensus conference of 1996. International

Journal of Oral Maxillofacial Implants 13S, 11–45.

Joyce, M.E., Jingushi, S., Bolander, M.E., 1990. Transforming growth factor beta in the regulation of fracture repair. The

Orthopedic Clinics of North America 21, 199–209.

Joyce, M.E., Jingushi, S., Scully, S.P., Bolander, M.E., 1991. Role of growth factors in in fracture healing. Progress in Clinical

and Biological Research 365, 391–416.

Lane, J.M., Tomin, E., Bostrom, M.P.G., 1999. Biosynthetic bone grafting. Clinical Orthopaedic and Related Research 367S,

107–117.

Lorenzetti, M., Mozzati, M., Campanino, P.P., Valente, G., 1998. Bone augmentation of the inferior floor of the maxillary sinus

with autogenous bone or composite bone grafts: a Histologic-histomorphometric preliminary report. International Journal of

Oral Maxillofacial Implants 13 (1), 69–76.

Montesinos, M.C., Desai, A., Chen, J.-F., Yee, H., Schwarzschild, M.A., Fink, J.S., Cronstein, B.N., 2002. Adenosine promotes

wound healing and mediates angigenesis in response to tissue injury via occupancy of A2A receptors. American Journal of

Pathology 160, 2009–2018.

Muratore, O., Pesce Schito, A., Cattarini, G., Tonoli, E.L., Gianoglio, S., Schiappacasse, S., Felli, L., Picchetta, F., Schito, G.G.,

1997. Evaluation of the trophic effect of human placental polydeoxyribonucleotides on human knee skin fibroblasts in

primary culture. Cellular Molecular Life Science 53, 279–285.

Musk, P., Campbell, R., Staples, J., Moss, D., Parson, P.G., 1989. Solar and UV induced mutation in human cells and inhibition

by deoxyribonucleotids. Mutatation Research 227, 25–30.

Nakamura, E., Uezono, Y., Narusawa, K., Shibuya, I., Oishi, Y., Tanaka, M., Yanaggihara, N., Nakamura, T., Izumi, F., 2000.

ATP activates DNA synthesis by acting on P2X receptors in human osteoblasts like MG-63 cells. American Journal of

Physiology (Cell Physiology) 279 (2), C510–C519.

Ostrom, R.S., Gregorian, C., Insel, P.A., 2000. Cellular release of and response to ATP as key determinants of set point of signal

transduction pathways. Journal of Biological Chemistry 16, 11735–11739.

Rathbone, M.P., Christjanson, L., De Forge, S., Deluca, B., Gysbers, J.W., Hindley, S., Jovetich, M., Middlemiss, P., Takal, S.,

1992a. Extracellular purine nucleosides stimulate cell division and morphogenesis: pathological and physiological impli-

cations. Medical Hypothesis 37, 232–240.

Rathbone, M.P., De Forge, S., Deluca, B., Gabel, B., Laurenssen, C., Middlemiss, P., Parkinson, S., 1992b. Purinergic

stimulation of cell division and differentiation: mechanism and pharmacological implications. Medical Hypothesis 37,

213–219.

Rathbone, M.P., Middlemiss, P.J., Gysbers, J.W., De Forge, S., Costello, P., Del Maestro, R.F., 1992c. Purine nucleosides and

nucleotides stimulate proliferation of a wide range of cells in vitro. In Vitro Cellular & Developmental Biology. Journal of

the Tissue Culture Association 28A, 529–536.

Reddi, A.H., 1992. Regulation of cartilage and bone differentiation by bone morphogenetic proteins. Current Opinion in

Cellular Biology 4, 850–855.

Service, R.F., 2000. Tissue engineers build new bone. Science 289, 1497–1500.

Siggelkow, H., Hilmes, D., Rebenstorff, K., Kurre, W., Huefner, M., 1998. Analysis of human primary bone cells by fluo-

rescence activated cell scanning: methodological problems and preliminary results. Clinica Chimica Acta; International

Journal of Clinical Chemistry 272, 111–125.

Sini, P., Denti, A., Cattarini, G., Daglio, M., Tira, M.E., Balduini, C., 1999. Effect of polydeoxyribonucleotides on human

fibroblasts in primary culture. Cell Biochemistry and Function 17, 107–114.

Thellung, S., Florio, T., Maragliano, A., Cattarini, G., Schettini, G., 1999. Polydeoxyribonucleotides enhance the proliferation

of human skin fibroblasts: involvement of A2 purinergic receptor subtypes. Life Sciences 64 (18), 1661–1674.

Tonello, G., Daglio, M., Zaccarelli, N., Sottofattori, E., Mazzei, B., Balbi, A., 1996. Characterization and quantitation of the

S. Guizzardi et al. / Life Sciences 73 (2003) 1973–1983 1983

active polynucleotide fraction (PDRN) from human placenta, a tissue repair stimulating agent. Journal of Pharmaceutical

and Biomedical Analysis 14, 1555–1560.

Urist, M.R., 1965. Bone: formation by autoinduction. Science 150, 893–899.

Urist, M.R., Strates, B.S., 1971. Bone morphogenetic protein. Journal of Dental Research 50S, 1392–1406.

Vega, C., Desai, A., Montesinos, M.C., Cronstein, B.N., 2002. Adenosine A2A receptor agonists promote more rapid wound

healng than recombinant human platelet derived growth factor (Beclaplermin Gel). Inflammation 26 (1), 19–24.

Viassov, V.V., Balakireva, L.A., Yabukov, L.A., 1994. Transport of oligonucleotides across natural and model membranes.

Biochimica et Biophysica Acta 1197, 95–108.