Quantification of Thrombocyte Growth Factors in Platelet Concentrates Produced by Discontinuous Cell Separation

Growth Factors, 2002 Vol. 20 (2), pp. 93 97 Quantification of Thrombocyte Growth Factors in Platelet Concentrates Produced by Discontinuous Cell Separation GERNOT WEIBRICH a, *, RAINER S.R. BUCH a, WILFRIED K.G. KLEIS a, GERD HAFNER b, WALTER E. HITZLER c and WILFRIED WAGNER a a Clinic for Oral and Maxillofacial Surgery, Johannes Gutenberg University of Mainz, Augustusplatz 2, D-55131 Mainz, Germany; b Institute of Clinical Chemistry and Laboratory Medicine, Johannes Gutenberg University of Mainz, Langenbeckstr. 1, D-55101 Mainz, Germany; c Transfusion Center, Johannes Gutenberg University of Mainz, Augustusplatz, D-55131 Mainz, Germany (Received 28 August 2001; Revised 11 April 2002) Platelet concentrates (PC) are increasingly used to increase bone regeneration in pre-prosthetic surgery. Although it is generally appreciated that certain growth factors (PDGF, TGF, EGF, and ECGF) are present in thrombocyte preparations, relatively little is known about these components in quantitative terms. The study reported here analysed the amounts of growth factors in PC produced under standard conditions from healthy volunteers. All the blood samples (237 in total) were analysed using Quantikine ELISA kits (R and D). The mean ^ SD platelet count in whole blood from these donors was 262; 000 ^ 58; 000=ml; while in PC produced by discontinuous cell separation it was 1,419,000 ^ 333,000/ml. The mean growth factor concentrations in PC preparations in ng/ml were as follows: PDGF-AB 125 ^ 55 ng/ml; TGF-b1 221 ^ 92 ng/ml; IGF-I 85 ^ 25 ng/ml; PDGF-BB 14 ^ 9 ng/ml; TGF-b2 0.4 ^ 0.3 ng/ml. These growth factor concentrations typically covered a 3 10 fold range: PDGF-AB 29 277 ng/ml; PDGF-BB 2 33 ng/ml; TGF-b1 32 397 ng/ml; TGF-b2 0.1 1.2 ng/ml; IGF-I 40 138 ng/ml. Platelet counts in PC were slightly higher for women (Mann Whitney Test all p, 0:001) than for men, while the concentrations of growth factors in PC exhibited no gender-related difference of any statistical significance. Keywords: Transforming growth factor; Platelet-derived growth factor; Insulin-like growth factor; Platelet concentrate; Platelet-rich plasma; Discontinuous cell separation INTRODUCTION Over the last decade, a number of growth factors have been identified and characterised. Endogenous growth factors also help to orchestrate bone regeneration, and it is well documented that the use of recombinant growth factors can be therapeutic influence (Rutherford et al., 1992; Park et al., 1995; Howell et al., 1997). There is also, commensurate interest in the optimised use of autologous growth factors produced after appropriate augmentation techniques. Platelets contain a number of different growth factors, including platelet-derived growth factor (PDGF), transforming growth factor beta 1 (TGF-b1), transforming growth factor beta 2 (TGF-b2), epidermal growth factor (EGF), and epithelial cell growth factor (ECFG), as well as a growth factor for hepatocytes (Kiuru et al., 1991). Marx et al. showed a statistically significant increase in bone formation and bone density after using thrombocyte growth factors supplied by platelet concentrates (PC), which these workers called platelet rich plasma (Marx et al., 1998). The added PC consisted of vital intact platelets, which were activated directly before the application by combination with calcium and bovine thrombine. Recently, the use of such PC to accelerate the osseo-integration of anosseous dental implants has been discussed (Rutherford et al., 1992; Anitua, 1999). Despite the increasing number of indications for the use of the mentioned PC in pre-prosthetic surgery, basic data on the normal values of the growth factor contents of these PC are not yet available. Possible influences of patient age and gender also remains unclear. Platelet count and growth factor content of PC likely depend on the particular technique used to obtain the PC. In addition, the donor s biological condition may be a determining factor in the composition of PC and its observed biological effects. Therefore, this study quantitatively assessed growth factor levels in PC produced *Corresponding author. Tel.: þ49-6131-17-3083. Fax: þ49-6131-17-6602. E-mail: weibrich@mkg.klinik.uni-mainz.de ISSN 0897-7194 q 2002 Taylor & Francis Ltd DOI: 10.1080/08977190290031950

94 G. WEIBRICH et al. by standard means from healthy donors, and considered the possible influences of the donor s whole blood thrombocyte count, age, and gender, on the PC preparation. Because it was not possible to quantify all of the multiple thrombocyte growth factors, three exemplary growth factors (TGF-b, PDGF, IGF), were investigated. The combination of TGF-b and PDGF has shown to strongly simulate the proliferation of osteoblast like cells in vitro (Lind, 1998). All three of them are considered to have a major effect on bone regeneration in vivo, as well in humans (Marx et al., 1998) as in clinical related animal models (Beck et al., 1991; Lynch et al., 1991). MATERIALS AND METHODS Between 20th December 2000 and 27th June 2001, 237 blood samples were collected from healthy donors (158 men, 79 women) aged between 21 and 62 years (mean 37, median 36, S.D. 11) at the Johannes Gutenberg University Transfusion Center (JGUTC). Before the platelets were separated, 50 ml of whole blood were drawn for serologic analysis via a cannula already in place. Subsequently, JGUTC prepared approximately 300 ml of platelet concentrate, using the discontinuous cell separation method described below. All donors had thrombocyte counts. 150,000/ml, as required by JGUTC s criteria for platelet donation. PC samples were stored in Eppendorf tubes at 2788C. The samples were thawed and centrifuged for 10 min at 10,000 rpm in a microcentrifuge immediately before assay at room temperature (RT). Each of the 237 specimens was analysed using commercial enzyme-linked immunosorbent assay kits (Quantikine ELISA kits, R and D Diagnostics, Wiesbaden, Germany) to quantify the concentrations of PDGF-AB (#DHD00), PDGF-BB (#DBB00), TGF-b1 (#DB100), TGF-b2 (#DB250), and IGF-I (#DG10). All growth factor assays were performed on specimens that had been frozen and stored. Deep freezing is a common method of releasing intracellular thrombocyte growth factors (Pesonen et al., 1989; Ito et al., 1993). Sekido et al. have shown that freezing specimens does not affect the levels of biologically active PDGF (Sekido et al., 1987). The quantitative ELISAs were performed twice according to the manufacturer s instructions. All quantitative measurements were described using summary statistics (n, mean, standard deviation, median, minimum, maximum, and other quantiles). Scatter plots and Pearson s correlation coefficient, r p, were used to demonstrate the relationship between whole blood, PC thrombocyte count, and growth factor content. In the case of non-normality and to analyse the influence of age, Spearman s rank correlation was used instead. To evaluate possible influences of gender, a Mann Whitney Test was performed on platelet counts and on growth factor levels in the samples. Production of Platelet Concentrates by Continuous Cell Separation PC was obtained by means of a Haemonetics gradient density cell separator (MCS 3p, Haemonetics, Munich, Germany) in routine use at JGUTC. This cell separator withdraws 400 450 ml of whole blood through a venous catheter, using intermittent (discontinuous) flow. The donor blood is supplemented with an anticoagulant (1 ml of citrate phosphate dextrose per 5 ml of blood) and allowed to flow into a rotating centrifuge cup, which separates the cellular blood components into erythrocyte, buffy coat (mostly thrombocytes and some leukocytes), and plasma (containing relatively few cells) fractions. As the centrifuge cup is refilled, the individual fractions leave the cup through automatic pressure valves and enter three separate bags. Some blood is allowed to recirculate into the centrifuge cup; the platelet recovery rate is higher for this portion because it remains in the centrifuge chamber for a longer period. After the erythrocyte and plasma fractions are re-transfused back to the donor, the separation process continues until a predefined volume of PC has been collected. The PC and venous blood samples thus obtained were subjected to automated platelet count analysis. RESULTS The mean platelet count in whole blood from the donors, in this study, was 262,000 ^ 58,000/ml (Fig. 1). The mean platelet count in the PC produced by JGUTC was 1,419,000 ^ 333,000/ml. Spearman s correlation coefficient was r s ¼ 0:747 for platelets in whole blood vs. PC. Three growth factors constituted the principal growth factors in the PC: PDGF-AB 125 ^ 55 ng/ml; TGF-b 221 ^ 92 ng/ml; and IGF-I 85 ^ 25 ng/ml. PDGF-BB 14 ^ 9 ng/ml and TGF-b2 0.4 ^ 0.3 ng/ml were present FIGURE 1 Thrombocyte counts in donors whole blood and platelet concentrate (PC) samples. Females ðn ¼ 79Þ have a slightly higher platelet count in PC compared to males ðn ¼ 158Þ: Extreme values are marked by circles.

THROMBOCYTE GROWTH FACTORS 95 FIGURE 2 Growth factor levels in platelet concentrate (PC) samples ðn ¼ 237Þ; presented in ng/ml. TGF-b1 and PDGF-AB are the dominant growth factors. Extreme values are marked by circles. only in small amounts (Fig. 2). The ranges for the different growth factors from the donor samples were as follows: PDGF-AB 29 277 ng/ml; PDGF-BB 2 33 ng/ml; TGF-b1 32 397 ng/ml; TGF-b2 0.1 1.2 ng/ml; IGF-I 40 138 ng/ml. Summary statistics defining the normal values for the growth factor content of the platelet concentrate derived by cell separation from healthy donors (including the 2.5 and 97.5 percentiles of the five growth factors assessed, and their confidence intervals of 90 and 95%, respectively) and the respective growth factor levels per 100,000 platelets are given in Table I. Pearson s and Spearman s correlation coefficients for whole blood platelets, and the respective PC platelets and growth factor content demonstrated little, if any, relationship between these parameters (all r p and r s, 0:5). On the other hand, the levels of PDGF-AB, PDGF-BB, and TGF-b1 exhibited relevant correlations. The levels of PDGF-AB were correlated with those for PDGF-BB ðr s ¼ 0:638Þ and TGF-b1 ðr s ¼ 0:600Þ; and levels of PDGF-BB were correlated with those for TGF-b1 ðr s ¼ 0:611Þ: The platelet concentrations in whole blood and PC were slightly higher for women ðn ¼ 79Þ than for men ðn ¼ 158Þ (Mann Whitney Test for gender p, 0:001 for both whole blood and PC). The mean genderdependent difference for platelet count was 30,430/ml for whole blood and 215,230/ml for PC. The growth factor concentrations in PC showed no gender-related difference of statistical significance. Age seemed to have no relevant influence on platelet counts or growth factor contents (all r s, 0:500; except for IGF r s ¼ 20:545). DISCUSSION The platelet counts in the PC obtained by JGUTC using discontinuous cell separation were in a range that TABLE I Descriptive statistical parameters for growth factor contents in platelet concentrates Percentile 97.5 (upper borderline of normal value) Percentile 2.5 (lower borderline of normal value) n Mean 95% Confidence interval Standard deviation Minimum Maximum Platelets in whole blood [1000/ml] 237 262 255 270 58 164 464 Platelets in PRP [1000/ml] 237 1419 1377 1462 333 21 2716 Leukocytes in PRP [/ml] 228 174 123 224 391 11 3530 PDGF-AB [ng/ml] 237 125 118 132 55 13 302 29 277 PDGF-BB [ng/ml] 237 14 13 15 9 1 49 2 33 TGF-b1 [ng/ml] 237 221 209 232 92 2 436 32 397 TGF-b2 [ng/ml] 237 0.4 0.4 0.5 0.3 0.04 1.7 0.1 1.2 IGF-I [ng/ml] 237 85 82 88 25 31 171 40 138 PGDF-AB [pg/100,000 thrombocytes] 237 9 8 10 8 1 109 PGDF-BB [pg/100,000 thrombocytes] 237 1 0.95 1.1 0.7 0.1 5.7 TGF-b1 [pg/100,000 thrombocytes] 237 16 15 17 6 0.1 50 TGF-b2 [pg/100,000 thrombocytes] 237 0.03 0.03 0.03 0.02 0.00 0.23 IGF [pg/100,000 thrombocytes] 237 8 5 11 22 2 341

96 G. WEIBRICH et al. FIGURE 3 PDGF-AB growth factor levels for washed platelets in five different dilution s (presented for three different donors) seem to evaluate a nearly linear correlation. corresponds to literature values (Marx et al., 1998; Menitove, 1999). The measured correlation between the baseline (donor whole blood) and end (PC) thrombocyte counts was slightly above 0.7 ðr s ¼ 0:747Þ: We assume that this result is due to isolated incidents of inefficient concentration. The levels of PDGFs in PC might be expected to depend on the number of platelets involved. There is not much literature data about PDGF levels in platelets. Singh et al. (1982) found 7.5 10 25 pg PDGF per platelet. Heldin et al. (1981) isolated 0.5 mg PDGF out of 3 10 13 platelets. There is no data about the amounts of the different PDGF sub-fractions (AA, AB and BB). Jiang et al. found a TGF-b level of 17.8 ^ 8.8 ng/10 5 platelets, which was much higher than the content of TGF-b1 and TGF-b2, which was analysed in this study ðn ¼ 237Þ (Jiang et al., 1995). Jiang et al. have used an ELISA test as well, but were using an antibody against three TGF-b sub-fractions (TGF-b1 þ 2 þ 3) for the growth factor analysis for his donor collective ðn ¼ 20Þ: Our data did not show a statistically significant correlation between platelet count and growth factor levels. This result might be explained by high between individual variability in cellular production or storage of cytokines. In the literature, a wide range of growth factor levels has already been described. Platelet concentrate contained 36.3 ^ 7.7 pg EGF/ml (after total thrombocyte lysis via six freeze/thaw cycles (Ito et al., 1993) and 38 505 pg VEGF/ml (after thrombocyte lysis with CaCl 2 or thrombin (Banks et al., 1998)). The growth factor analysis for washed thrombocytes of three different healthy donors in five different concentrations seems to have a good linear correlation (Figs. 3 and 4), which means that the scattering of growth factor concentrations FIGURE 4 TGF-b1 growth factor levels for washed platelets in five different dilutions (presented for three different donors) seem to evaluate a nearly linear correlation. for an individual patient seems to be limited. Another cause for the scattering of the growth factor results that might be discussed can be seen in the induction of the variability by the freeze thaw centrifuge method. It seems improbable, that this is a major cause, because the conditions used are quite harsh (2788C and 10,000 rpm microcentrifuge spin) and because the dilution series was done with five different aliquots for each of the three donors, which were frozen, thawed and centrifuged separately and evaluated a linear relation between platelet and growth factor concentration for the individual donors. Unfortunately, the data obtained in this study demonstrate that there is no simple procedure available for obtaining pre-operative estimates of individual growth factor levels in a PC sample. Neither whole blood nor PC platelet counts are predictive for the resulting growth factor levels in PC. Predictive estimates for some growth factors are possible by analysing the PGDF-AB content, but are restricted as well. It might be expected that the platelet growth factors in the PC lead to an improved bone regeneration for different reasons: (1) It is known that platelets have a major function during the physiological healing process of a bone fracture. (2) PC contain platelets in a 5-fold concentration compared to the whole blood. For this reason, it can be expected that there is a much higher platelet and growth factor concentration in the resulting clot. (3) Cell structures have shown a concentration dependent increase in the proliferation rate of human osteoblast like cells. (4) The synergic stimulative effect of the combination of various growth factors on osteoblasts in vitro has already been described in the literature

THROMBOCYTE GROWTH FACTORS 97 (Lind, 1998). On the other side, platelets are a biological reservoir for a physiological mixture of a great variety of different growth factors. CONCLUSIONS The platelet count of PC produced by discontinuous cell separation is sufficiently predictable from the platelet count of whole blood. The resulting PC typically contain PDGF-AB, TGF-b1, and IGF-I in high concentrations and PDGF-BB and TGF-b2 in low concentrations, but individual samples exhibit high variation between different donors. Because of the great variety of different growth factors in platelets and their 5-fold concentration in PC an associative positive biological effect on the bone regeneration in vivo by the use of PC can be assumed. Acknowledgements The authors thank Ms A.H. Loos of the Institute for Medical Statistics and Documentation at the Johannes Gutenberg University of Mainz for help with statistical analyses. References Anitua, E. (1999) Plasma rich in growth factors: preliminary results of use in the preparation of future sites for implants, Int. J. Oral Maxillofac. Implants 14(4), 529 535. Banks, R.E., Forbes, M.A., Kinsey, S.E., Stanley, A., Ingham, E., Walters, C. and Selby, P.J. (1998) Release of the angiogenic cytokine vascular endothelial growth factor (VEGF) from platelets: significance for VEGF measurements and cancer biology [see comments], Br. J. Cancer 77(6), 956 964. Beck, L.S., Deguzman, L., Lee, W.P., Xu, Y., McFatridge, L.A., Gillett, N.A. and Amento, E.P. (1991) TGF-beta1 induces bone closure of skull defects, J. Bone Miner. Res. 6(11), 1257 1265. Heldin, C.H., Westermark, B. and Wasteson, A. (1981) Platelet-derived growth factor. Isolation by a large-scale procedure and analysis of subunit composition, Biochem. J. 193(3), 907 913. Howell, T.H., Fiorellini, J.P., Paquette, D.W., Offenbacher, S., Giannobile, W.V. and Lynch, S.E. (1997) A phase I/II clinical trial to evaluate a combination of recombinant human platelet-derived growth factor-bb and recombinant human insulin-like growth factor-i in patients with periodontal disease, J. Periodontol. 68(12), 1186 1193. Ito, J., Kamiya, Y. and Kawaguchi, M. (1993) Increased content of epidermal growth factor in platelet lysates in non-insulin-dependent diabetes mellitus, Life Sci. 53(9), 717 724. Jiang, X., Kanai, H., Hiromura, K., Sawamura, M. and Yano, S. (1995) Increased intraplatelet and urinary transforming growth factor-beta in patients with multiple myeloma, Acta Haematol. 94(1), 1 6. Kiuru, J., Viinikka, L., Myllyla, G., Pesonen, K. and Perheentupa, J. (1991) Cytoskeleton-dependent release of human platelet epidermal growth factor, Life Sci. 49(26), 1997 2003. Lind, M. (1998) Growth factor stimulation of bone healing. Effects on osteoblasts, osteotomies, and implants fixation, Acta Orthop. Scand. Suppl. 69, 1 37. Lynch, S.E., Buser, D., Hernandez, R.A., Weber, H.P., Stich, H., Fox, C.H. and Williams, R.C. (1991) Effects of the platelet-derived growth factor/insulin-like growth factor-i combination on bone regeneration around titanium dental implants. Results of a pilot study in beagle dogs, J. Periodontol. 62(11), 710 716. Marx, R.E., Carlson, E.R., Eichstaedt, R.M., Schimmele, S.R., Strauss, J.E. and Georgeff, K.R. (1998) Platelet-rich plasma: Growth factor enhancement for bone grafts, Oral Surg. Oral Med. Oral Pathol. Oral Radiol. Endod. 85(6), 638 646. Menitove, J.E. (1999) Standards for Blood Banks and Transfusion Services (American Association of Blood Banks, Bethesda, MD). Park, J.B., Matsuura, M., Han, K.Y., Norderyd, O., Lin, W.L., Genco, R.J. and Cho, M.I. (1995) Periodontal regeneration in class III furcation defects of beagle dogs using guided tissue regenerative therapy with platelet-derived growth factor, J. Periodontol. 66(6), 462 477. Pesonen, K., Viinikka, L., Myllyla, G., Kiuru, J. and Perheentupa, J. (1989) Characterization of material with epidermal growth factor immunoreactivity in human serum and platelets, J. Clin. Endocrinol. Metab. 68(2), 486 491. Rutherford, R.B., Niekrash, C.E., Kennedy, J.E. and Charette, M.F. (1992) Platelet-derived and insulin-like growth factors stimulate regeneration of periodontal attachment in monkeys, J. Periodontal Res. 27(4 Pt 1), 285 290. Sekido, Y., Morishima, Y. and Ohya, K. (1987) Activity of plateletderived growth factor (PDGF) in platelet concentrates and cryopreserved platelets determined by PDGF bioassay, Vox Sang. 52(1 2), 27 30. Singh, J.P., Chaikin, M.A. and Stiles, C.D. (1982) Phylogenetic analysis of platelet-derived growth factor by radio-receptor assay, J. Cell Biol. 95(2 Pt 1), 667 671.